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CA 02625349 2008-04-07
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METHODS AND COMPOSITIONS RELATING TO ANTHRAX SPORE
GLYCOPROTEINS AS VACCINES
STATEMENT OF RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent Application
60/724,306, filed October 6, 2005, and entitled "Novel Anthrax Spore Vaccine."
FIELD OF THE INVENTION
The present invention relates methods and compositions relating to anthrax
spore
glycoproteins as vaccines.
BACKGROUND
Anthrax was previously known as woolsorters' disease as human infection had
usually
resulted from contact with infected animals or animal products such as hides
or wool. The
events of September 11, 2001 and the subsequent anthrax outbreaks highlighted
the more recent
use of this bacterium for biological warfare and terrorism. Louis Pasteur
produced the first
anthrax vaccine in 1881 using a heat attenuated strain. The current U.S.
licensed human anthrax
vaccine, BIOTHR.AXTM or Anthrax Vaccine Adsorbed (AVA) produced by BioPort
Corporation
(Lansing, MI), consists of aluminum hydroxide-adsorbed supernatant material
from fermentor
cultures of a toxigenic, non-encapsulated strain of B. anthracis.
Only toxin components have thus far been shown to confer protective immunity
agaifist
anthrax (Mahlandt, B. G., et al. 1966. J Immuno196:727-33). For example,
protective antigen
(PA) is an essential component of an anthrax vaccine (Grabenstein, J. D. 2003,
Inimunol.
Allergy Clin. North Am., 23(4):7I3-30). Anti-PA antibody specific immunity may
include anti-
spore activity and thus, may have a role in impeding the early stages of
infection with B.
anthracis spores (Wellcos, S. et al., 2001, Microbiology 147:1677-85). The
currentU.S. licensed
human anthrax vaccine, primarily consists of protective antigen (PA) and
undefined quantities of
Lethal Factor (LF) and Edema Factor (EF), from fermentor cultures of a
toxigenic, non-
encapsulated strain of B. anthracis. Human vaccination with BIOTHRAXTM may
require six
immunizations followed by annual boosters (2002, Anthrax Vaccine Adsorbed
(BioThraxTM)
Product Insert, BioPort Corporation; Friedlander, A. M., et a1.,1999, Jama
282:2104-6). Using
this vaccine, about 1 percent systemic and 3.6 percent local adverse events in
humans have been
reported (Pitlinan, P. R. et al., 2001, Vaccine 20:972-8).
CA 02625349 2008-04-07
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There have been many attempts to improve the safety profile and immunogenicity
of the
anthrax vaccine using PA as an antigen, including the formulation of PA in
adjuvants (Ivins, B.
E. et al., 1992, Infect. Immun., 60:662-8; Kenney, R. T., et al., 2004. J.
Infect. Dis., 190:774-82,
Epub 2004 Jul 13) (Matyas, G. R., et al., 2004, Infect. Iinmun., 72:1181-3),
conjugating capsular
poly-gamma-d-glutamic acid (PGA) to PA (Rhie, G. E. et al., 2003. Proc. Natl.
Acad. Sci., USA
100:10925-30), the use of purified PA (Singh, Y. et al., 1998. Infect. Immun.,
66:3447-8) and C-
domain 4 of PA (PA-D4), (Flick-Smith, H. C. et al., 2002, Infect. hnmun.,
70:1653-6), the
development of PA-based DNA vaccines (Gu, M. L. et al., 1999, Vaccine 17:340-
4;
Riemenschneider, J. et al., 2003, Vaccine 21:4071-80), and expression of PA in
adenovirus,
Salmonella typhinaurium, Bacillus subtilis, vaccinia viral vector, and
venezuelan equine
encephalitis virus (Coulson, N. M. et al. ,1994, Vaccine, 12:1395-401;
Garmory, H. S. et al.,
2003, Infect. Immun., 71:3831-6; Iacono-Connors, L. C. et al., 1991, Infect.
Immun., 59:1961-5;
Ivins, B. E., and S. L. Welkos, 1986, Infect. Immun., 54:537-42; Lee, J. S. et
al., 2003., Infect.
Immun., 71:1491-6; Tan, Y. et al. 2003, Hum. Gene Ther., 14:1673-82). Anthrax
protective
antigen (PA) is the major antigen in the current licensed antlirax vaccine
BIOTHRAXTM. The c-
terminal domain 4 (PA-D4, residues 596-735) of PA appears to be responsible
for binding
cellular receptor, the anthrax toxin receptor (ATR), and may contain the
dominant protective
epitopes of PA (Fliclc-Smith, H. C. et al., 2002, Infect. Immun. 70:1653-6;
Little, S. F. et al.
1996, Microbiology 142:707-15). Previous research indicated that immunization
witll plasmid
expression vectors in a combination of PA and N-temiinal region truncated LF
(residues 10-254
of the mature protein) may provide better protection than PA alone (Galloway,
D., et al. 2004,
Vaccine, 22:1604-8; Price, B. M. et al., 2001, Infect. Immun., 69:4509-15).
The highly fatal nature of pulmonary anthrax, the ease of production and
storage of the
spores of B. anthr acis, aiid the ability of spores to survive in the
environment after an attack,
malce B. antlzracis attractive as an agent in biowarfare and bioterrorism.
Because the window of
opportunity for effective antibiotic treatinent is so small, vaccination may
be the best defense
against pulmonary anthrax. The current vaccine against anthrax is a crude
culture supematant
from a non-encapsulated strain ofB. anthracis that contains protective antigen
(PA) generated by
the vegetative cell. This vaccine may provide protection against the pulmonary
form of anthrax
in rhesus macaques and rabbits, but protection in guinea pigs is variable
(Fellows et al., 2001).
Furthermore, the current vaccine which utilizes PA can only be expected to
afford protection
against the natural agent, and would not be expected to provide protection
against engineered
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forms of the organism. The selection of B. aiatlaYacis as a biological weapon
is due not only to
the toxic properties of the bacterium, but also because it provides an easily
produced, stably
maintained, delivery veliicle. It is possible to introduce other toxins, such
as botulism toxin or
shiga toxin, into this bacterium. Such engineered B. ayathracis spores could
then deliver not only
the anthrax toxin, but also the additional toxins introduced into the spore.
The current vaccine
(which utilizes PA) would not be effective against such engineered organisms
because it provides
no protection against the foreign toxins. For these reasons, antitoxin
immunity alone may not be
a long-term solution.
While the currently available vaccines are an improvement over the use of a
heat-
attenuated anthrax strain, there is still a need for an improved vaccine. For
exainple, the currently
available vaccines are characterized by a lack of standardization, and a
relatively high expense of
production. Additionally, human vaccination with BIOTHRAXTM requires six
immunizations
followed by annual boosters (see e.g., the Anthrax Vaccine Adsorbed BIOTHRAXTM
Product
Insert, BioPort Corporation, 2002; Friedlander, A. M., et al., 1999, JAMA
282:2104-6). Further
underscoring the need for development of new, improved anthrax vaccines are
the reported 1%
systemic and 3.6% local adverse events in humans (Pittman, P. R. et al., 2001,
Vaccine 20:972-
8).
Thus, there is a need to provide methods and systems for the isolation pof
porteins
complexes from the surface of microorganisms, where such complexes may be
antigenic. There
is also a need to develop vaccines that may be used to defend against various
biowarfare agents
as well as other disease agents such as HIV.
SUMMARY OF THE INVENTION
Embodiments of the present invention comprise methods and compositions
relating to
isolation of glycoprotein complexes from anthrax and other microbiological
agents for use as
vaccines. The present invention may be embodied in a variety of ways.
In one embodiment, the present invention comprises a inethod for isolation of
glycoproteins on the exosporium or surface of a microorganism that inay be
used in a vaccine. In
an embodiment, the microorganism may be Bacillus arathracis or an anthrax-
lilce bacterim. In an
enibodiment, the method may comprise the step of isolating at least one
glycoprotein from an
extract of the exosporium of the bacterium by absorption of the extract to a
sugar-binding agent.
In an einbodiment, the sugar binding agent is lectin. Or, otlier agents such
as proteins, lipids,
sugars and other antibodies that can combine with sugars, and that are lcnown
to interact with
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specific sugars found in glyoproteins may be used to capture proteins and
other glycoprotein
complexes.
In another embodiment, the present invention coinprises a composition
comprising at
least one glycoprotein isolated from the exosporium or surface of a
microorganism, where the
glycoprotein comprises at least one lectin-binding sugar. In an embodiinent,
exosporium is froin
an Bacillus afatlaracis spore. In an embodiment, the coinposition may comprise
a pharmaceutical
carrier. In certain embodiments the glycoprotein is isolated as a complex
comprising at least one
of an oligosaccharide, a lipid, or a phospholipid.
In certain embodiments, the compositions of the present invention provide an
anthrax
vaccine that is protective against all strains Bacillus anthracis or
associated diseases, and other
anthrax-like infections including, but not liinited to, Bacillus cereus G9241.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention may be better understood by reference to the following
non-limiting
drawings.
FIG. 1 illustrates a schematic presentation of the exosporium of the Bacillus
anthracis
spore in accordance with an embodiment of the present invention.
FIG. 2 illustrates a flow-chart presentation of a method for the isolation of
glycoproteins
from the exosporium of the Bacillus anthracis spore in accordance with an
embodiment of the
present invention.
FIG. 3 illustrates an embodimeiit of protein distribution of Bacillus
anthracis spores
before and after lectin treatment run by one-dimensional gel electrophoresis
in accordance with
an embodiment of the present invention.
FIG. 4 illustrates glycoprotein staingi of urea extracted spores before lectin
treatment run
by two dimensiorial gel electorphoresis in accordance with an embodinient of
the present
invention.
FIG. 5 illustrates a MALDI TOF MS characterization of a single glycoprotein
band (EAl
1D) (band 1 of the gel of FIG. 3) in accordance with an einbodiment of the
present invention.
DETAILED DESCRIPTION
Definitions
The following definitions may be used to understand the description herein.
Unless
defined otherwise, all teclmical and scientific terms used herein have the
same meaning as
commonly understood by one of ordinary slcill in the art.
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The term "a" or "an" as used herein may refer to mo're than one object unless
the
context clearly indicates otherwise. The term "or" is used interchangeably
with the term
"and/or" unless the context clearly indicates otherwise.
"Polypeptide" and "protein" are used interchangeably herein to describe
protein
molecules that may comprise either partial or full-length proteins. As used
herein, a
"polypeptide domain" comprises a region along a polypeptide that comprises an
independent
unit. Domains may be defined in terms of structure, sequence and/or biological
activity. In
one embodiment, a polypeptide domain may comprise a region of a protein that
folds in a
manner that is substantially independent from the rest of the protein. Domains
may be
identified using domain databases such as, but not limited to PFAM, PRODOM,
PROSITE,
BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS. As used
herein, the term "glycoprotein" refers to any protein that is glycosylated.
A "nucleic acid" is a polynucleotide such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA). The term is used to include single-stranded nucleic
acids, double-
stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside
analogues.
DNA molecules may be identified by their nucleic acid sequences , which are
generally
presented in the 5' to 3' direction (as the coding strand), where the 5' and
3' indicate the
linkages formed between the 5'-hydroxyl group of one nucleotide and the 3'-
hydroxyl group
of the next nucleotide. For a coding strand presented in the 5'-3' direction,
its complement
(or non-coding strand) is the DNA strand which hybridizes to that sequence
according to
Watson-Crick base pairing. Thus, as used herein, the complement of a nucleic
acid is the
same as the "reverse complement" and describes the nucleic acid that in its
natural form,
would be based paired with the nucleic acid in question.
As used herein, "primers" are a subset of oligonucleotides that can hybridize
with a target
nucleic acid such that an enzymatic reactions, that uses the primers as a
substrate, at least in part,
can occur. A primer can be made from any combination of nucleotides or
nucleotide derivatives
or analogs available in the art which do not interfere with the enzymatic
manipulation. "Probes"
are oligonucleotide molecules capable of interacting with a target nucleic
acid, typically in a
sequence specific manner, for example through hybridization. Typically a probe
can be made
from any combination of nucleotides or nucleotide derivatives or analogs
available in the art.
The tenn "vector" refers to a nucleic acid molecule that inay be used to
transport a second
nucleic acid molecule into a cell. In one embodiment, the vector allows for
replication of DNA
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sequences inserted into the vector. The vector may comprise a promoter to
enhance expression
of the nucleic acid molecule in at least some host cells. Vectors may
replicate autonomously
(extrachromasomal) or may be integrated into a host cell chromosome. In one
embodiment, the
vector may comprise an expression vector capable of producing a protein
derived from at least
part of a nucleic acid sequence inserted into the vector.
The term "percent identical" or "percent identity" refers to sequence identity
between
two amino acid sequences or between two nucleic acid sequences. Percent
identity can be
determined by aligning two sequences and refers to the number of identical
residues (i.e.,
amino acid or nucleotide) at positions shared by the compared sequences.
Sequence
alignment and comparison may be conducted using the algorithms standard in the
art (e.g.
Smith and Waterman, Adv. Appl. Matla., 1981, 2:482; Needleman and Wunsch,
1970, J: Mol.
Biol., 48:443); Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444)
or by
computerized versions of these algorithms (Wisconsin Genetics Software Package
Release
7.0, Genetics Coinputer Group, 575 Science Drive, Madison, WI) publicly
available as '
BLAST and FASTA. Also, ENTREZ, available through the National Institutes of
Health,
Bethesda MD, may be used for sequence comparison. In one embodiment, percent
identity of
two sequences may be determined using GCG with a gap weight of 1, such that
each amino
acid gap is weighted as if it were a single ainino acid or nucleotide mismatch
between the two
sequences.
An "effective amount" as used herein means the amount of an agent that is
effective for
producing a desired effect. Where the agent is being used to achieve a
insecticidal effect, the
actual dose which comprises the effective amount may depend upon the route of
administration,
and the formulation being used.
As used herein, an "immune response" refers to reaction of the body as a whole
to the
presence of an antigen which includes malcing antibodies, developing
unnlunity, developing
hypersensitivity to the antigen, and developing tolerance. Therefore, an
immune response to an
antigen also includes the development in a subject of a humoral and/or
cellular immune response
to the antigen of interest. A "humoral immune response" is mediated by
antibodies produced by
plasma cells. A "cellular immune response" is one mediated by T lymphocytes
and/or other
white blood cells. Spores can germinate within macrophages, so immunization to
a spore can
cause the development of opsonizing antibodies. Cell niediated iminunity can
compensate by
causing macrophage activation and increased spore death. Humoral immunity to
spore
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components can also cause immunity, and this effect may be augmented by cell
mediated
immunity. As used herein, "antibody titers" are defined as the highest
dihition in post-immune
sera that resulted in equal absorbance value of pre-iinmune samples for each
subject.
As used herein, the term "antigen" refers to any agent, (e.g.., any substance,
compound,
molecule, protein or other moiety) that is recognized by an antibody and/or
can elicit an immune
response in an individual. As used herein, the term "adjuvant" refers to any
agent (e.g., any
substance, compound, molecule, protein or other moiety) that can increase the
immune response
of an antigen.
As used herein, the term "antibody" encompasses, but is not limited to, whole
immunoglobulin (i.e., an intact antibody) of, any class. Native antibodies are
usually
heterotetrameric glycoproteins, composed of two identical light (L) chains and
two identical
heavy (H) chains. Typically, each light chain is linlced to a heavy chain by
one covalent disulfide
bond, while the number of disulfide linlcages varies between the heavy chains
of different
immunoglobulin isotypes. Each heavy and light chain may also have regularly
spaced intrachain
disulfide bridges. Each heavy chain may have at one end a variable domain VH
followed by a
number of constant domains. Each light chain may have a variable domain at one
end VL and a
constant domain at its other end; the constant domain of the light chain may
be aligned with the
first constant domain of the heavy chain, and the light chain variable domain
may be aligned with
the variable domain of the heavy chain. Particular amino acid residues are
believed to fomi an
interface between the light and heavy chain variable domains. The light chains
of antibodies
from any vertebrate species can be assigned to one of two clearly distinct
types, called kappa (x)
and lambda (X), based on the amino acid sequences of their constant domains.
Depending on the
amino acid sequence of the constant domain of their heavy chains,
immunoglobulins can be
assigned to different classes. There are five major classes of human
immunoglobulins: IgA, IgD,
IgE, IgG and IgM, and several of these may be further divided into subclasses
(isotypes), e.g.,
IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. There are similar class for
other species (e.g.,
mouse). The heavy chain constant domains that correspond to the different
classes of
immunoglobulins are called alpha, delta, epsilon, gamma, and inu,
respectively.
The term "variable" is used herein to describe certain portions of the
variable antibody
domains that differ in sequence among antibodies and are used in the binding
and specificity of
each particular antibody for its particular antigen. However, the variability
is not usually evenly
distributed througli the variable domains of antibodies, but is typically
concentrated in three
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segments called complementarity determining regions (CDRs) or hypervariable
regions both in
the light chain and the heavy chain variable domains. The more highly
conserved portions of the
variable domains are called the frameworlc (FR). The variable domains of
native heavy and light
chains each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by
three CDRs, which can form loops connecting, and in some cases forming part
of, the b-sheet
structure. The CDRs in each chain may be held together in close proximity by
the FR regions
and, with the CbRs from the other chain, contribute to the formation of the
antigen binding site
of antibodies (see Kabat E. A. et al., 1987, "Sequences of Proteins of
Immunological Interest,"
National Institutes of Health, Bethesda, Md.). The constant domains are not
involved directly in
binding an antibody to an antigen, but may exhibit various effector functions,
such as
participation of the antibody in antibody-dependent cellular toxicity.
As used herein, the term "antibody or fragments thereof' encompasses chimeric
antibodies and hybrid antibodies, with dual or multiple antigen or epitope
specificities, and
fragments, such as F(ab')2, Fab', Fab and the like, including hybrid
fraginents. Thus, fragments
of the antibodies that retain the ability to bind their specific antigens are
included in this
definition. For example, fragments of antibodies which maintain EFn binding
activity are
included within the meaning of the temi "antibody or fragment thereof." Such
antibodies and
fragments can be made by techniques known in the art and can be screened for
specificity and
activity according to the methods set fortll in the Examples and in general
methods for producing
antibodies and screening antibodies for specificity and activity (See Harlow
and Lane.
Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New Yorlc,
(1988)). Also
included within the meaning of "antibody or fragments thereof' are conjugates
of antibody
fragments and antigen binding proteins (single chain antibodies) as described,
for example, in
U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by
reference.
Also, as used herein, "huinanized forms of antibodies" are chimeric
immunoglobulins,
iminunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2,
or other antigen-
binding subsequences of antibodies) which contain minimal sequence derived
from non-human
immunoglobulin.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a
substantially homogeneous population of antibodies, i.e., the individual
antibodies coinprising
the population are identical except for possible naturally occurring
inutations that may be present
in minor amounts. The monoclonal antibodies herein specifically include
"chinieric" antibodies
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in which a portion of the heavy and/or liglit chain is identical with or
homologous to
corresponding sequences in antibodies derived from a particular species or
belonging to a
particular antibody class or subclass, while the remainder of the chain(s) is
identical with or
homologous to corresponding sequences in antibodies derived from another
species or belonging
to another antibody class or subclass, as well as fragments of such
antibodies, so long as they
exhibit the desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et
al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)).
As used herein, the term "anthrax" refers to any strain of Bacillus anthracis
either in
vegatative or spore form. As used herein, the terms "anthrax-lilce" or
"anthrax-lilce infections" or
"anthrax-lilce diseases" refer to any strain of Bacillus cereus or other
related Bacillus strain, and
diseases similar to that of inhalation, gastrointestinal, or cutaneous
anthrax. As used herein, the
term "spore surface" refers to the exosporium, spore coat, and the outer layer
of the cortex.
Specifically, B. cereus ATCC 10987, B. cereus ATCC 10987, B. cereus G9241 have
been known
to cause anthrax-like response in recent studies. (Rask et al., 2004, Nucleic
Acids Res. 32(3):977-
88; Han et al., 2006; J. Bacteriology, 188 (9): 3382-90; Hoffinaster et al.,
2006, J Clin.
Microbiol., 44: 3352-60).
As used herein, the term "complexed," "complex," or "complexes" means anything
that is
bound together by eithe covalent or non-covalent interactions. For example,
the glycoprotein
BcIA complex is Bc1A and any other proteins, lipids, phospholipids,
polysaccharides or
glycoproteins bound to BclA.
Methods And Compositions Relating To Anthrax Spore Glycoproteins As Vaccines
Embodiments of the present invention comprise methods and coinpositions
relating to the
isolation anthrax spore glycoproteins and glycoprotein complexes as vaccines.
The present
invention may be enzbodied in a variety of ways.
In one embodiment, the present invention comprises a method for isolation of
glycoproteins on the exosporium of a microorganism that may be used in a
vaccine. In am
embodiment, the microorganism may be a bacterium. In an embodiment, the
bacteriuin may be
Bacillus antlaracis or an anthrax-like bacterium. In an embodiment, the method
may comprise
the step of isolating at least one glycoprotein from an extract of the
exosporium of the bacterium
by absorption of the extract to a sugar-binding agent. In an embodiment, the
sugar binding agent
is lectin. Or, other agents, such as proteins, lipids, sugars and other
antibodies that are laiown to
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interact with specific sugars found in glyoproteins may be used to capture
glycoproteins or
glycoprotein complexes.
In an embodiment, the method comprises a step wherein the glycoprotein is
isolated as
part of a complex comprising at least one other molecule, wllerein the at
least one other
molecule comprises a protein, an oligosaccharide, a lipid, or a phospholipid.
For example,
the complex may be isolated from the exosporium using at least one of size-
exclusion
chromatography or electro-elution. Or other size selection method may be used.
Also, in an
embodiment, at least one other molecule of the complex is identified. In an
embodiment, the
methods used to identify the glycoprotein and/or other molecule may include MS-
TOF,
protein sequencing or other similar methods such as Matrix-assisted laser
desorption/ionization (MALDI), Time-of-flight (TOF) mass spectrometry (MS),
Electrospray-
ionization (ESI) Ion Trap (IT) MS,
Matrix-assisted laser desorption/ionization (MALDI) Fourier transfonn ion
cyclotron
resonance (FT-ICR) MS, Electrospray ionization (ESI) Fourier transform ion
cyclotron
resonance (FT-ICR) MS.
For example, in one embodiment, the present invention comprises a method for
isolation
of glycoproteins on the exosporium of the Bacillus anthf acis spore that may
be used in a
vaccine. In an embodiment, the method may coinprise the step of isolating at
least one
glycoprotein from an extract of the exosporium of the Bacillus antlaracis
spore by absorption of
proteins in the extract to lectin. In certain embodiments, the glycoprotein is
isolated as a complex
comprising at least one of an oligosaccharide, a lipid, or a phospholipid.
In an embodiment, the glycoprotein is isolated as part of a complex comprising
at
least one other, molecule, wherein the at least one other molecule comprises a
protein, an
oligosaccharide, a lipid, or a phospholipid. For example, the coinplex may be
isolated from
the exosporium using at least one of size-exclusion cliroinatography or
electro-elution. Or
other size selection method may be used. Also, in an embodiment, at least one
other
molecule of the complex is identified. In an embodiment, the methods used to
identify the
glycoprotein and/or other molecule may include MS-TOF, protein sequencing or
other similar
methods such as Matrix-assisted laser desorption/ionization (MALDI), Time-of-
flight (TOF)
mass spectrometry (MS), Electrospray-ionization (ESI) Ion Trap (IT) MS, Matrix-
assisted
laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance
(FT-ICR)
MS, Electrospray ionization (ESI) Fourier transfonn ion cyclotron resonance
(FT-ICR) MS.
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In an embodiment, the complex comprises at least one of the following proteins
from
Bacillus aratlaracis: CotS, CotJA, CotJB, CotJC, CotM, CotH, CotC , CotAlpha,
CotF, CotD,
CotZ, Cot(Putative 1, 2, 3, 4), CotHypoAlpha, CotE, CotF(Related), Bc1A, EA1,
EA2, srtA
(Sortase A), SSPH1, SSPH2, SSPI, SSPK, SSPN, SSPO, TLP, SSPB, SSPalpha/betal,
SSPalpha/beta2, SSPalpha/beta3, SSPalpha/beta4, SASP-2, SSPF, SASP-l,
SSPE(SSPgamma),
ExsB, cspA, cspB-l, cspB-2, cspC, cspD, cspE, NDK, NupC-1, NupC-2, NupC-3,
NupC-4,
NupC-5, NupC-6, NupC-7, PnuC, Alanine racemase, Alanine dehydrogenase,
Nucleoside
hydrolase, BxpB, ExsFA, or ExsFB.
In another einbodiment, the coinplex is isolated from a Bacillus subtilis
spore. Thus, in
an embodiment, the complex comprises at least one of the following proteins
from Bacillus
subtilis: CotA, CotB, CotC, CotD, CotE, CotF, CotG, CotH, CotJA, CotJB, CotJC,
CotM, CotR,
CotSA, CotS, CotT, CotV, CotW, CotY, CotZ, GerPA, GerPB, GerPC, GerPD, GerPE,
GerPF,
YaaH, YabG, YrbA (SafA), CotQ (YvdP), CotU (YnzH), CotI (YtaA), YckK, YdhD,
YhdA,
YhdE, YirY, YisY, Yodl, YopQ, YdeP/YpeB, YpzA, YusA, YwqH, YxeF, CspD, Hsb,
PhoA,
SleB, SspA, SspE, YhcN, YrbB, CggR, CoxA, CwIJ, SpoIVA, SpoVM, SpoVID, YhbA,
CSI5,
CspB, CspC, CspD, DHBA, FABI, RL10, SRFAD, SAS 1, SAS2, SASG, SSPA, SSPB,
SSPC,
SSPD, SSPE, SSPF, SSPG, SSPH, SSPI, SSPJ, SSPK, SSPL, SSPM, SSPN, SSPO, SSPP,
TLP,
SSPG-1, or SSPG-2.
In another embodiment, the complex is isolated from a Bacillus cereus spore.
Thus, in an
embodiment, the complex comprises at least one of the following proteins from
Bacillus cereus:
ExsA, ExsB, ExsC, ExsD, ExsE, ExsG, ExsH, ExsY, ExsJ, ExsF, YrbB, or NadA.
In another embodiment, the present invention comprises a composition
comprising at
least one glycoprotein from the exosporium of the Bacillus aratlaracis spore,
where the
glycoprotein comprises at least one lectin-binding sugar. In certain
embodiments the
glycoprotein is isolated as a complex comprising at least one of an
oligosaccharide, a lipid, or a
phospholipid. In an embodiment, the coinposition may comprise a
phannaceutically acceptable
carrier.
Pharmaceutically acceptable carriers may comprise any of the standard
pharmaceutically
accepted carriers lrnown in the art. In one embodiment, the pharmaceutical
carrier may be a
liquid and the protein or nucleic acid construct of the present invention may
be in the form of a
solution. In anotlzer embodinient, the phannaceutically acceptable carrier may
be a solid in the
form of a powder, a lyophilized powder, or a tablet. Or, the phannaceutical
carrier maybe a gel,
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suppository, or cream. In alternate embodiments, the carrier may comprise a
liposome, a
microcapsule, a polymer encapsulated cell, or a virus. Thus, the term
phannaceutically
acceptable carrier encompasses, but is not limited to, any of the standard
pharmaceutically
accepted carriers, such as water, alcohols, phosphate buffered saline
solution, sugars (e.g.,
sucrose or mannitol), oils or emulsions such as oil/water emulsions or a
trigyceride emulsion,
various types of wetting agents, tablets, coated tablets and capsules.
In an embodiment, the complex comprises at least one of the following proteins
from
Bacillus asathf=acis: CotS, CotJA, CotJB, CotJC, CotM, CotH, CotC , CotAipha,
CotF, CotD,
CotZ, Cot(Putative 1, 2, 3, 4), CotHypoAlpha, CotE, CotF(Related), Bc1A, EA1,
EA2, srtA
(Sortase A), SSPH1, SSPH2, SSPI, SSPK, SSPN, SSPO, TLP, SSPB, SSPalpha/betal,
SSPalpha/beta2, SSPalpha/beta3, SSPalpha/beta4, SASP-2, SSPF, SASP-1,
SSPE(SSPgainma),
ExsB, cspA, cspB-1, cspB-2, cspC, cspD, cspE,,NDK, NupC-1, NupC-2, NupC-3,
NupC-4,
NupC-5, NupC-6, NupC-7, PnuC, Alanine racemase, Alanine dehydrogenase,
Nucleoside
hydrolase, BxpB, ExsFA, or ExsFB.
In another embodiment, the complex is isolated from a Bacillus subtilis spore.
Thus, in
an embodiment, the complex coinprises at least one of the following proteins
from Bacillus
subtilis: CotA, CotB, CotC, CotD, CotE, CotF, CotG, CotH, CotJA, CotJB, CotJC,
CotM, CotR,
CotSA, CotS, CotT, CotV, CotW, CotY, CotZ, GerPA, GerPB, GerPC, GerPD, GerPE,
GerPF,
YaaH, YabG, YrbA (SafA), CotQ (YvdP), CotU (YnzH), Cotl (YtaA), YckK, YdhD,
YhdA,
YhdE, YirY, YisY, Yodl, YopQ, YdeP/YpeB, YpzA, YusA, YwqH, YxeF, CspD, Hsb,
PhoA,
SIeB, SspA, SspE, YhcN, YrbB, CggR, CoxA, Cw1J, SpoIVA, SpoVM, SpoVID, YhbA,
CSI5,
CspB, CspC, CspD, DHBA, FABI, RL10, SRFAD, SAS1, SAS2, SASG, SSPA, SSPB, SSPC,
SSPD, SSPE, SSPF, SSPG, SSPH, SSPI, SSPJ, SSPK, SSPL, SSPM, SSPN, SSPO, SSPP,
TLP,
SSPG-1, or SSPG-2.
In another embodiment, the complex is isolated from a Bacillus cereus spore.
Thus, in an
embodiment, the complex comprises at least one of the following proteins from
Bacillus cereus:
ExsA, ExsB, ExsC, ExsD, ExsE, ExsG, ExsH, ExsY, ExsJ, ExsF, YrbB, or NadA.
In an einbodiment, the method comprises a step wlierein the glycoprotein is
isolated as part
of a complex coniprising at least one other molecule, wherein the at least one
other molecule
comprises a protein, an oligosaccharide, a lipid, or a phospholipid. For
exaniple, the coinplex
may be isolated from the exosporium using at least one of size-exclusion
cliromatography or
electro-elution. Or otlier size selection method may be used. Also, in an
einbodiment, at least
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one other molecule of the complex is identified. In an embodiment, the methods
used to
identify the glycoprotein and/or other molecule may include MS-TOF, protein
sequencing or
other similar metliods such as Matrix-assisted laser desorption/ionization
(MALDI), Time-of-
flight (TOF) mass spectrometry (MS), Electrospray-ionization (ESI) Ion Trap
(IT) MS,
Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion
cyclotron
resonance (FT-ICR) MS, Electrospray ionization (ESI) Fourier transform ion
cyclotron
resonance (FT-ICR) MS.
In yet other embodiments, the present invention comprises compositions
coinprising a
complex isolated from the exosporium of the Bacillus anthracis spore
coinprising at least one of
a polypeptide, glycoprotein, lipid, phospholipid, or oligosaccharide wherein
the polypeptide,
glycoprotein, lipid, phospholipids, or oligosaccharide coinprises an antigen,
and/or wherein the at
least one polypeptide, glycoprotein, lipid, phospholipid, or oligosaccharide
is capable of
producing a cellular or a humoral immune response. In an embodiment, the
composition may
comprise a phamiaceutically acceptable carrier.
In an embodiment, the complex comprises at least one of the following proteins
from
Bacillus ari.tlai=acis: CotS, CotJA, CotJB, CotJC, CotM, CotH, CotC ,
CotAlpha, CotF, CotD,
CotZ, Cot(Putative 1, 2, 3, 4), CotHypoAlpha, CotE, CotF(Related), Bc1A, EA1,
EA2, srtA
(Sortase A), SSPH1, SSPH2, SSPI, SSPK, SSPN, SSPO, TLP, SSPB, SSPalpha/betal,
SSPalpha/beta2, SSPalpha/beta3, SSPalpha/beta4, SASP-2, SSPF, SASP-1,
SSPE(SSPgamma),
ExsB, cspA, espB-1, cspB-2, cspC, cspD, cspE, NDK, NupC-1, NupC-2, NupC-3,
NupC-4,
NupC-5, NupC-6, NupC-7, PnuC, Alanine racemase, Alanine dehydrogenase,
Nucleoside
liydrolase, BxpB, ExsFA, or ExsFB.
In another embodiment, the complex is isolated from a Bacillus subtilis spore.
Thus, in
an embodiment, the complex comprises at least one of the following proteins
from Bacillus
subtilis: CotA, CotB, CotC, CotD, CotE, CotF, CotG, CotH, CotJA, CotJB, CotJC,
CotM, CotR,
CotSA, CotS, CotT, CotV, CotW, CotY, CotZ, GerPA, GerPB, GerPC, GerPD, GerPE,
GerPF,
YaaH, YabG, YrbA (SafA), CotQ (YvdP), CotU (YnzH), CotI (YtaA), YckK, YdhD,
YhdA,
YhdE, YirY, YisY, Yodl, YopQ, YdeP/YpeB, YpzA, YusA, YwqH, YxeF, CspD, Hsb,
PhoA,
S1eB, SspA, SspE, YhcN, YrbB, CggR, CoxA, CwlJ, SpoIVA, SpoVM, SpoVID, YhbA,
CSI5,
CspB, CspC, CspD, DHBA, FABI, RL10, SRFAD, SAS1, SAS2, SASG, SSPA, SSPB, SSPC,
SSPD, SSPE, SSPF, SSPG, SSPH, SSPI, SSPJ, SSPK, SSPL, SSPM, SSPN, SSPO, SSPP,
TLP,
SSPG-1, or SSPG-2.
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WO 2007/044607 PCT/US2006/039293
In another embodiment, the complex is isolated from a Bacillus cereus spore.
Thus, in an
embodiment, the complex comprises at least one of the following proteins from
Bacillus cereus:
ExsA, ExsB, ExsC, ExsD, ExsE, ExsG, ExsH, ExsY, ExsJ, ExsF, YrbB, or NadA.
In an embodiment, the glycoprotein is isolated as part of a complex comprising
at
least one other molecule, wherein the at least one other molecule comprises a
protein, an
oligosaccharide, a lipid, or a phospholipid. For example, the complex may be
isolated from
the exosporium using at least one of size-exclusion chromatography or electro-
elution. Or
other size selection method may be used. Also, in an embodiment, at least one
other
molecule of the complex is identified. In an embodinient, the methods used to
identify the
glycoprotein and/or other molecule may include MS=TOF, protein sequencing or
other similar
methods such as Matrix-assisted laser desorption/ionization (MALDI), Time-of-
flight (TOF)
mass spectrometry (MS), Electrospray-ionization (ESI) Ion Trap (IT) MS,
Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion
cyclotron
resonance (FT-ICR) MS, Electrospray ionization (ESI) Fourier transform ion
cyclotron
resonance (FT-ICR) MS.
In an embodiment, the microorganism from which the glycoprotein or
glycoprotein
complex is isolated may comprise an Anthrax bacterium. Or, other the
microorganims may
comprise any one of the microorganisms listed in Table 1.
Table 1
Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
FEBS Letters, vol. 217,
no. 2, pp. 145-157,
Escherichia coli 171cDa Man 1987 1987
Arch Biochem Biophys
2001 Jun 1;390(1):109-
Escherichia coli 181cDa Gal 2001 18
Arch Biochem Biophys
2001 Jun 1;390(1):109-
Escherichia coli 18 kDa Gal 2001 18
Infection and Immunity.
Streptococcus 1996 Sep; 64(9): 3659-
suis 18-kDa Gal(al-4)Gal 1996 65
20-1cDa Infect. Immun., 1996
Escherichia coli subunits GIcNAc 1996 Jan;64(1):332-42
Infection and Immunity,
Burlcholderia vol. 64, no. 4, pp. 1420-
cepacia 22-kDa Gal(al-4)Gal 1996 1425, 1996
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Pasteurella Glycobiology, 2000,
haemolytica 68-kDa G1cNAc 2000 Vol. 10, No. 1 31-37
Pasteurella Glycobiology, 2000,
haemolytica 68-kDa NeuAc 2000 Vol. 10, No. 1 31-37
Gal(b 1-3)[NeuAc(a2-
Clostridium 3)]Ga1NAc(b 1-4)Gal(b 1-
botulinum type B 4)[NeuAc(a2-3)Glc(b1- Microbial Pathogenesis.
neurotoxin B subunit 1)Cer 1998 1998 Aug 25(2): 91-9
The Journal of
Experimental Medicine.
1986 Jun 1 163(6):
Shiga toxin B subunit Gal(al-3)Gal(bl-4)Glc 1986 1391-404
The Journal of
Experimental Medicine.
Gal(al-3)Gal(bl- 1986 Jun 1 163(6):
Shiga toxin B subunit 4)G1cNAc 1986 1391-404
The Journal of
Experimental Medicine.
1986 Jun 1 163(6):
Shiga toxin B subunit G1cNAc(bi-4)G1cNAc 1986 1391-404
B- Journal of Immunology.
Ricin toxin subunit (bl-3)Gal 2004 2004; 172: 6836-6845
B- Journal of Immunology.
Ricin toxin subunit (bl-4)Gal 2004 2004; 172: 6836-6845
Biochemical and
B- Biophysical Research
subunit; Gal(bl-3)Ga1NAc(bl- Communications.2004
Cholera toxin pentameri 4)[NeuAc(a2-3)]Gal(b 1- Aug 13; vol. 321, no.1:
(Vibrio cholerae) c 4)Glc(bl-1) 2004 192-196
Biochemical and
B- Biophysical Research
subunit; NeuAc(a2-3)[Gal(bl- Conununications.2004
Cholera toxin pentameri 3)GalNAc(bl-4)]Gal(bl- Aug 13; vol. 321, no.1:
(Vibrio cholerae) c 4)Glc(bl-1) 2004 192-196
Fuc(al-2)[Gal(al-
Helicobacter 3)Gal(bl- Science. 2004 Ju123;
pylori BabA 3)]G1cNAc[Fuc(al-4)] 2004 Vo1305:519-22
Fuc(a l -2) [Ga1NAc(a 1-
Helicobacter 3)Gal(bl-3)]Fuc(al- Science. 2004 Ju123;
pylori BabA 4)[G1cNAc] 2004 Vo1305:519-22
Helicobacter Fuc(al-2)[Ga1NAc(al- Science. 2004 Ju123;
pylori BabA 3)Gal(bl-3)]G1cNAc 2004 Vo1305:519-22
Fuc(a1-2)[Ga1NAc(a 1-
Helicobacter 3)Gal(bl- Science. 2004 Ju123;
pylori BabA 3)]GlcNAc[Fuc(al-4)] 2004 Vo1305:519-22
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Helicobacter Fuc(al-2)Gal(bl- Science. 2004 Ju123;
pylori BabA 3)Fuc(al-4)[G1cNAc] 2004 Vol 305: 519-22
Helicobacter Fuc(al-2)Gal(bl- Science. 2004 Ju123;
pylori BabA 3)G1cNAc 2004 Vo1305:519-22
Gal(al-3)Gal(bl-
Helicobacter 3)[Fuc(al-2)]Fuc(al- Science. 2004 Ju123;
pylori BabA 4)[GIcNAc] 2004 Vol 305: 519-22
Gal(al-3)Gal(bl-
Helicobacter 3)[Fuc(al- Science. 2004 Ju123;
pylori BabA 2)]G1cNAc[Fuc(al-4)] 2004 Vol 305: 519-22
Ga1NAc(al-3)Gal(b 1-
Helicobacter 3)[Fuc(al-2)]Fuc(al- Science. 2004 Ju123;
pylori BabA 4)[GlcNAc] 2004 Vo1305:519-22
Helicobacter Ga1NAc(al-3)Gal(bl- Science. 2004 Ju123;
pylori BabA 3)[Fuc(al-2)]G1cNAc 2004 Vo1305:519-22
Ga1NAc(al-3)Gal(bl-
Helicobacter 3)[Fuc(al- Science. 2004 Ju123;
pylori BabA 2)]GlcNAc[Fuc(al-4)] 2004 Vo1305:519-22
Int J Med Microbiol.
Ga1NAc(bl-4)[NeuGc(al- 2000 Mar;290(1):27-
Escherichia coli CfaB 3)]Gal(bl-4)Glc(bl-1)Cer 2000 35. Review
hit J Med Microbiol.
NeuGc(al-3)[Ga1NAc(bl- 2000 Mar;290(1):27-
Escherichia coli CfaB 4)]Gal(bl-4)Glc(bl-1)Cer 2000 35. Review
Journal of
Microbiological
Methods. Vol. 34, no.
1, pp. 23-29. 1 Sep
Escherichia coli Class I G Gal(al-4)Gal 1998 1998
Journal of
Microbiological
Methods. Vol. 34, no.
Class II 1, pp. 23-29. 1 Sep
Escherichia coli G Gal(al-4)Gal 1998 1998
Journal of
Microbiological
Methods. Vol. 34, no.
Class III 1, pp. 23-29. 1 Sep
Escherichia coli G Gal(al-4)Gal 1998 1998
Infection and Immunity,
vol. 63, no. 2, pp. 640-
Escherichia coli CS3 Ga1NAc(bl-4)Gal 1995 646, 1995
Pseudomonas exoenzym Gal(bl-3)GaINAc(bl- Gene. 1997 Jun 11;
aeruginosa e S 4)Gal(bl-4)Glc(bl-1)Cer 1997 192(1): 99-108
Pseudomonas exoenzym Ga1NAc(bl-4)Gal(bl- Gene. 1997 Jun 11;
aeruginosa e S 4)Glc(bl-l)Cer 1997 192(1): 99-108
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Journal of
Microbiological
Methods. Vol. 34, no.
1, pp. 23-29. 1 Sep
Escherichia coli F Gal(al-4)Gal 1998 1998
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia eoli FaeG Fuc 2000 35. Review
Int J Med Microbiol.
2000 Mar;290(l):27-
Escherichia coli FaeG Gal(b 2000 35. Review
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia coli FaeG Gal(bl-3)Gal 2000 35. Review
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia coli FaeG Ga1NAc 2000 35. Review
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia coli FaeG G1cNAc 2000 35. Review
Int J Med Microbiol.
NeuGc(al-3)Gal(bl- 2000 Mar;290(l):27-
Escherichia coli FanC 4)Glc(bl-l)Cer 2000 35. Review
Bordetella Gal(b1-3)G1cNAc(b1- Infection and Immunity.
pertussis FHA 3)Gal(bl-4)Glc(bl-1)Cer 1993 1993 Jul; 61(7): 2780-5
Emerg Infect Dis. 1999
May-Jun;5(3):395-403.
Escherichia coli FimH Man 1999 Review
J. Bacteriol., Febraary
15, 1999; 181(4): 1059
Escherichia coli FimH Man 1999 - 1071
Molecular
microbiology, 2002
Escherichia coli FimH Man 2002 May, 44(4):903-15
Med Sci Monit. 2003
Escherichia coli FimH Man 2003 Mar;9(3):RA76-82
Int J Med Microbiol.
Escherichia coli FocH Gal 2000 2000 Mar;290(l):27-35
Int J Med Microbiol.
Escherichia coli FocH Ga1NAc 2000 2000 Mar;290(1):27-35
Human PNAS of the United
Immunodeficiency States of America. 1993
Virus gp120 Gal(bl-1)Cer 1993 Apr 1; 90(7): 2700-4
Heavy Infection and Inununity.
Entamoeba (170-1cDa) Vol. 65, no. 5, pp.
histolytica subunit Gal 1999 2096-2102. May 1999
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Heavy Infection and Immunity.
Entamoeba (170-kDa) Vol. 65, no. 5, pp.
histolytica subunit Ga1NAc 1999 2096-2102. May 1999
Gal(al-3)Gal(bl-
4)G1cNAc(b l -
6)[NeuAc(a2-3)Gal(b 1-
4)Glc(b l-3)]Gal(b l- Biochem Pharmacol.
hemagglu 4)G1cNAc(bl-3)Gal(b1- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
NeuAc(a2-3)[NeuAc(a2- Biochem Pharmacol.
hemagglu 3)Gal(bl-3)Ga1NAc(bl- 2003 Mar 1;65(5):699-
Influenza tinin 4)]Gal(bl-3)Glc(b1-1) 2003 707. Review
NeuAc(a2-3 )Gal(b 1-
3)Ga1NAc(b 1- Biochem Pharmacol.
hemagglu 4)[NeuAc(a2-3)]Gal(b1- 2003 Mar 1;65(5):699-
Influenza tinin 3)Glc(bl-1) 2003 707. Review
Biochem Pharmacol.
hemagglu NeuAc(a2-3)Gal(bl- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
NeuAc(a2-3 )Gal(b 1-
4)Glc(b 1-3)[Gal(al-
3)Gal(bl-4)G1cNAc(b1- Biochem Pharmacol.
hemagglu 6)]Gal(bl-4)G1cNAc(b1- 2003 Mar 1;65(5):699-
Influenza tinin 3)Gal(bl-4)Glc(bl-1) 2003 707. Review
NeuAc(a2-3)Gal(b 1- Biochem Pharmacol.
hemagglu 4)G1cNAc(b1-4)Gal(b1- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
NeuAc(a2-3 )Gal(b 1-
4)G1cNAc(bl-4)Gal(bl- Biochem Pharmacol.
hemagglu 4)G1cNAc(b1-3)Gal(bl- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
NeuAc(a2-6)Gal(b 1- Biochem Pharmacol.
hemagglu 4)G1cNAc(bl-4)Gal(bl- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
Biochem Pharmacol.
hemagglu NeuGc(a2-3)Gal(bl- 2003 Mar 1;65(5):699-
Influenza tinin 4)Glc(bl-1) 2003 707. Review
Jounial of Virology.
hemagglu 1990 Oct; 64(10):
Rotavirus tinin NeuAc 1990 4830-5
Light (35-
or 31- Infection and Immunity.
Entamoeba kDa) Vol. 65, no. 5, pp.
histolytica subunit Gal 1999 2096-2102. May 1999
Light (35-
or 31- Infection and Immunity.
Entamoeba 1cDa) Vol. 65, no. 5, pp.
histolytica subunit Ga1NAc 1999 2096-2102. May 1999
Int J Med Microbiol.
2000 Mar;290(1):27-
Proteus mirabilis MrpII Gal(al-4)Gal 2000 35. Review
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year C'itation
NeuAc(a2-3)Gal(b 1-
3)[NeuAc(a2- Infect. Immun., August
6)]Ga1NAc(bl-3)Gal(al- 1, 1998; 66(8): 3856 -
Escherichia coli P 4)Gal(bl-4)Glc(bl-1)Cer 1998 3861
NeuAc(a2-3)Gal(bl- Infect. Immun., August
3)GaINAc(bl-3)Gal(al- 1, 1998; 66(8): 3856 -
Escherichia coli P 4)Gal(bl-4)Glc(bl-1)Cer 1998 3861
NeuAc(a2-6) [NeuAc(a2-
3)Gal(bl-3)]Ga1NAc(bl- Infect. Immun., August
3)Gal(al-4)Gal(bl- 1, 1998; 66(8): 3856 -
Escherichia coli P 4)Glc(bl-1)Cer 1998 3861
Pseudomonas , Microbes and Infection.
aeruginosa PA-IIL Fuc 2004 2004 Feb; 6(2): 221-8
Pseudomonas Microbes and Infection.
aeruginosa PA-IIL Man 2004 2004 Feb; 6(2): 221-8
Pseudomonas Microbes and Infection.
aeruginosa PA-IL Gal 2004 2004 Feb; 6(2): 221-8
Current Opinion in
Structural Biology, vol.
5, no. 5, pp. 622-635,
Escherichia coli PapG Gal(al-4)Gal 1995 1995
Emerg Infect Dis. 1999
May-Jun;5(3):395-403.
Escherichia coli PapG Gal(al-4)Gal 1999 Review
Infect. Immun.,
November 1, 1999;
Escherichia coli PapG Gal(al-4)Gal 1999 67(11): 6161 - 6163
J. Bacteriol., February
15, 1999; 181(4): 1059
Escherichia coli PapG Gal(al-4)Gal 1999 - 1071
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia coli PapG Gal(al-4)Gal 2000 35. Review
Med Sci Monit. 2003
Escherichia coli PapG Gal(al-4)Gal 2003 Mar;9(3):RA76-82
Bioorganic & medicinal
chemistry, 1996 Nov,
Escherichia coli PapG Gal(al-4)Gal(b 1996 4(11):1809-17
Ga1NAc(bl-3)Gal(al- EMBO Rep., July 1,
Escherichia coli PapGII 4)Gal(bl-4)Glc(bl-1)Cer 2001 2001; 2(7): 621 - 627
Ga1NAc(al-3)Ga1NAc(a1- EMBO Rep., July 1,
Escherichia coli PapGIII 3)Gal(al-4)Gal(bl-4)Cer 2001 2001; 2(7): 621 - 627
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Journal of
Microbiological
Methods. Vol. 34, no.
1, pp. 23-29. 1 Sep
Escherichia coli PapGJ96 Gal(al-4)Gal 1998 1998
Infection and Immunity.
1998 Sep; 66(9): 4545-
Yersinia pestis pH 6 Gal(bl-l)Cer 1998 8
Infection and Immunity.
Gal(bl-3)GaINAc(bl- 1998 Sep; 66(9): 4545-
Yersinia pestis pH 6 4)Gal(b1-4)Glc(bl-1)Cer 1998 8
Infection and Immunity.
1998 Sep; 66(9): 4545-
Yersinia pestis pH 6 Gal(bl-4)Glc(bl-1)Cer 1998 8
Infection and Immunity.
Ga1NAc(bl-4)Gal(bl- 1998 Sep; 66(9): 4545-
Yersinia pestis pH 6 4)Glc(bl-1)Cer 1998 8
Pseudomonas pilin Gal(bl-3)Ga1NAc(bl- Gene. 1997 Jun 11;
aeruginosa subunit 4)Gal(bl-4)Glc(bl-1)Cer 1997 192(1):99-108
Pseudomonas pilin Gene. 1997 Jun 11;
aeruginosa subunit Ga1NAc(bl-4)Gal 1997 192(1): 99-108
Pseudomonas pilin Ga1NAc(bl-4)Gal(bl- Gene. 1997 Jun 11;
aeruginosa subunit 4)Glc(b1-1)Cer 1997 192(1):99-108
The Journal of
biological chemistry.
Streptococcus Dec 1, 1995. v. 270
suis PN Gal(al-4)Gal 1995 (48) p. 28874-28878
The Journal of
biological chemistry.
Streptococcus Dec 1, 1995. v. 270
suis PO Gal(al-4)Gal 1995 (48) p. 28874-28878
Emerg Infect Dis. 1999
May-Jun;5(3):395-403.
Escherichia coli PrsG Gal(al-4)Gal 1999 Review
Int J Med Microbiol.
2000 Mar;290(1):27-
Escherichia coli PrsG Gal(al-4)Gal 2000 35. Review
Med Sci Monit. 2003
Escherichia coli PrsG Gal(al-4)Gal 2003 Mar;9(3):RA76-82
Pertussis toxin PNAS United States of
(Bordetella S2 Gal(bl-3)G1cNAc(bl- America. 1992 Jan 1;
pertussis) subunit 3)Gal(bl-4)Glc(bl-1)Cer 1992 89(1):118-22
Pertussis toxin PNAS United States of
(Bordetella S3 Gal(bl-3)Ga1NAc(bl- America. 1992 Jan 1;
pertussis) subunit 4)Gal(bl-4)Glc(bl-1)Cer 1992 89(1):118-22
Med Sci Monit. 2003
Escherichia coli SafS NeuAc(a2-3)Gal 2003 Mar;9(3):RA76-82
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Pathogen or
Toxin Lectin Carbohydrate or Ligand Year Citation
Emerg Infect Dis. 1999
Escherichia coli SfaS NeuAc(a2-3)Gal 1999 May-Jun;5(3):395-403
Int J Med Microbiol.
Escherichia coli SfaS NeuAc(a2-3)Gal 2000 2000 Mar;290(1):27-35
transmem
brane
heavy
subunit
(Hgl; 170 Infection and Immunity.
Entamoeba kDa) 2004 vol. 72, no. 9:
histolytica disulfide Gal 2004 5349-5357
transmem
brane
heavy
subunit
(Hgl; 170 Infection and Immunity.
Entamoeba kDa) 2004 vol. 72, no. 9:
histolytica disulfide G1cNAc 2004 5349-5357
Virus
Spike Journal of Virology,
Protein vol. 71, no. 9, pp. 6749-
Rotavirus VP4 NeuAc 1997 6756, Sep 1997
In an embodiment, the composition may comprise a vaccine. In certain
embodiments, the
compositions of the present invention provide an anthrax vaccine that is
protective against all
strains Bacillus anthracis, and other anthrax-like infections including, but
not limited to, Bacillus
cereus G924 1. The vaccines may comprise a purified antigen, wherein the
antigen comprises the
any one of the polypeptides disclosed lierein. In an embodiment, the antigen
may comprise a
complex of at least one glycoprotein isolated from the exosporium of a
Bacillus anthracis spore.
In certain embodiments, the vaccine may comprise a combination vaccine, where
the
combination vaccine comprises a purified antigen isolated from the exosporium
of a Bacillus
afathNacis spore, and another Bacillus anthf acis antigen, such as protective
antigen (PA), the
lethal factor (LF) protein, edema factor (EF), and the lilce.
In certain embodiments of the methods or compositions of the present
invention, the
complex comprises an isolated molecule comprising at least one of the nucleic
acid sequences or
at least one of the amino acid sequences, as set forth in SEQ ID NOs: 1-26.
Or, the complex may
comprise a nucleic acid molecule having 95%-99% identity to the nucleic acid
sequences, or a
protein or polypeptide having 95%-99% identity amino acid sequences, as set
forth in SEQ ID
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NOs: 1-26. In other embodiments, the complex may comprise a nucleic acid
molecule having
90%-99% identity to the nucleic acid sequences, or a protein or polypeptide
having 90%-99%
identity amino acid sequences, as set forth in SEQ ID NOs: 1-26. In other
embodiments, the
complex may comprise a nucleic acid molecule having 85%-99% identity to the
nucleic acid
sequences, or a protein or polypeptide having 85%-99% identity amino acid
sequences as set
forth in SEQ ID NOs: 1-26. In yet other embodiments, the complex may comprise
a nucleic acid
molecule having 80%-99% identity to the nucleic acid sequences, or a protein
or polypeptide
having 80%-99% identity amino acid sequences as set forth in SEQ ID NOs: 1-26.
For example,
the complex may comprise a fragment and/or homologue of a protein encoded by
at least one of
the nucleic acid and/or amino acid sequences, respectively, as set forth in
SEQ ID NOs: 1-26,
wherein the homologue comprises conseivative amino acid substitutions and the
fragment
comprises the portion of the polypeptide that is antigenic. The present
invention also comprises
fragments of nucleic acid sequences that comprise at least 15 consecutive
nucleic acid sequences
for the nucleic acid sequences included in the sequences as set forth in SEQ
ID NOs: 1-26. In yet
another embodiment, the present invention also comprises fragments of nucleic
acid sequences
that comprise at least 15 consecutive nucleic acid sequences for the
complement of nucleic acid
sequences included in the sequences as set forth in SEQ ID NOs: 1-26. In an
embodiment, the
glycoprotein comprises an amino acid sequence having at least 80% homology to
at least one of
the amino acid sequences as set forth in SEQ ID. NOs: 2, SEQ ID. NO: 4, SEQ
ID. NO: 6, SEQ
ID. NO: 8, SEQ ID. NO: 10, SEQ ID. NO: 12, SEQ ID. NO: 14, SEQ ID. NO: 16, SEQ
ID. NO:
18, SEQ ID. NO: 20, SEQ ID. NO: 22, SEQ ID. NO: 24, SEQ ID. NO: 26. For
example, in an
embodiment, the present invention comprises an isolated nucleic acid molecule
encoding a lectin-
binding glycoprotein isolated from the exosporium of the Bacillus anthracis
spore comprising a
nucleic acid sequence as set forth in SEQ ID NO: 1, SEQ ID. NO: 3, SEQ ID. NO:
5, SEQ ID.
NO: 7, SEQ ID. NO: 9, SEQ ID. NO: 11, SEQ ID. NO: 13, SEQ ID. NO: 15, SEQ ID.
NO: 17,
SEQ ID. NO: 19, SEQ ID. NO: 21, SEQ ID. NO: 23, or SEQ ID. NO: 25.
In an embodiment, the present invention also comprises vectors, wherein the
vectors
comprise recombinant DNA constructs comprising any of the nucleic acids
disclosed herein.
Also, the present invention may comprise cells comprising vectors that
comprise recombinant
DNA constructs coinprising any of the nucleic acids disclosed herein.
In yet another enibodiment, the present invention comprises methods of using
these
compositions for vaccination against anthrax infection and anthrax-lilce
infections such as
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Bacillus cereus G9241. For example, in an embodiment, the compositions of the
present
invention can be used, either alone or in combination, as an antigen for
eliciting protective
immunity against anthrax. In an embod'unent, the composition can be used witli
an adjuvant to
help elicit an immune response.
The present invention also provides methods of preventing or treating anthrax
infection.
In another embodiment, the present invention comprises a method of treating or
preventing
anthrax infection, anthrax-like diseases, or other diseases of interest in a
subject, comprising
administering to the subject a composition coinprising at least one
glycoprotein from the
exosporium of the Bacillus anthracis spore. Thus, in an elnbodiinent, the
present invention
comprises a method of producing an immune response to Bacillus anthracis in a
subject
comprising administering to the subject the composition comprising a
composition comprising at
least one glycoprotein on the exosporium of the Bacillus antlzracis spore,
where the glycoprotein
comprises at least one lectin-binding sugar. In an embodiment, the immune
response is a cellular
immune response. Alternatively or additionally, the iinmune response is a
humoral immune
response. In yet another embodiment, the present invention comprises a method
of producing an
immune response to Bacillus anthracis in a subject comprising administering to
the subject any
of the nucleic acids disclosed herein, whereby the nucleic acid of the
composition can be
expressed, for example, wherein the immune response is a cellular or humoral
immune response.
The subjects treated with the vaccines and compositions of the present
invention can be
any mammal, such as a mouse, a primate, a human, a bovine, an ovine, an
ungulate, or an equine.
The compositions and/or vaccines of the present invention can be administered
in any manner
standard to vaccine adnlinistration. In an embodiment, adniinistration is by
injection. In another
embodiment, administration may be by nasal inhalation.
The compositions and vaccines disclosed =herein can be used individually, or
in
combination witli other components of a spore from anthrax or an anthrax-like
bacterium. Or,
the compositions and vaccines may be used in combination with vaccines used to
treat antlirax
infection such as vaccines comprising protective antigen (PA), LF or EF
(Pezard, C. et al. 1995,
Infect. Immun., 63:1369-72) vaccine. Furthermore, the vaccines disclosed
herein may include the
use of an adjuvant. Also, otlier B. antlzracis antigens can may be used
(Brossier, F., and M.
Mock, 2001, Toxicol., 39:1747-55; Cohen, S et al., 2000, Infect Inimun 68:4549-
58).
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Anthrax and other anthrax like infections
Anthrax is a highly fatal disease primarily of cattle, sheep and goats caused
by the Gram-
positive, endospore-producing, rod-shaped bacterium Bacillus anthracis. B.
anthracis, like the
other members of the genus Bacillus, can shift to a developmental pathway,
sporulation, when
growth conditions become unfavorable. The result of the sporulation process is
the production of
an endospore, a metabolically inert form of the cell wllich is refractive to
numerous
environmental insults including desiccation and heat. The spores produced by
Bacillus species
can persist in soil for long periods of time and are found worldwide.
Humans are also susceptible to infections by B. anthracis. Infections can
occur in one of
three forms. Entry of spores through abrasions in the slcin results in the
production of a lesion
referred to as a malignant pustule, wliich is the hallmarlc of the cutaneous
form of anthrax. This
form is the most common form of "natural" human anthrax, has a low mortality
rate, and
responds well to antibiotic treatment. Ingestion of anthrax contaminated meat
gives rise to the
gastrointestinal form of the disease. This type of the disease is rare in the
United States, although
cases were reported in Minnesota in the year 2000 (Morbid. Mortal. Weekly
Report, 2000,
49:813-816). This form of the disease has a higher mortality rate,
approximately 40% in
untreated cases. The most lethal form of human anthrax is the pulmonary form.
Inhaled spores
are deposited in the lungs and are engulfed by the alveolar macrophages (Ross,
J. M. , 1957, J.
Pathol. Bacteriol, 73:485-494). The spores are then transported to the
regional lymph nodes,
germinating inside the macrophages en route (Ross, 1957; Guidi-Rontani, C.,
M., et al., 1999,
Mol. Microbiol. 31:9-17). The early symptoms of pulmonary anthrax are
nondescript influenza-
lilce syinptoms. The patient's condition deteriorates rapidly after the onset
of symptoins and death
often occurs within a few days. The mortality rate is high, 98% or greater,
even with antibiotic
therapy. Pulmonary anthrax is thus the primary concern in a bioterrorism
attack. Recently, a
strain of Bacillus cereus G9241 has been shown to cause a disease similar to
inhalation anthrax
(Hoffmaster, A.R., et al., 2004, Proc. Natl. Acad. Sci., USA, 101: 8449-8454).
In mice, B.
cereus G9241 is 100% lethal (Hoffinaster et al., 2004). Other strains of
cereus have shown some
of the virulence factors of B. anthf acis such as B. cereus ATCC 10987 (Rask
et al., 2004; Han et
al., 2006, and Hoffinaster et al., 2006). It may be possible to coinbat
infection from anthrax and
anthrax like diseases with a single vaccine.
The spore is the infectious form of B. anthracis. The outside of the spore is
characterized
by the presence of an external exosporium that consists of a basal layer
surrounded by an extenial
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nap of hair-like projections (Hoffinaster et al., 2004; Haclusuka, Y., et al.,
1966, J. Bacteriol.
91:2382-2384; Kramer, M. J., and I. L. Rotli, 1968, Can J. Microbiol. 14:1297-
1299). Upon
entry of spores in the lung, the spores are rapidly talcen up by
macrophages,where they germinate.
In the vegetative form (multiplicative form) the spore=exosporium and coat
layers are replaced by
a poly-D-glutamic acid capsule and S (surface) layers.
The fate of macrophage engulfed spores has been examined (Dixon, T. C., et
al., 2000,
Cell. Microbiol., 2:453-463; Guidi-Rontani, C., et al., 1999, Mol. Microbiol.
31:9-17; Guidi-
Rontani, C., et al., 2001, Molec. Microbiol. 42:931-938). When spores of B.
anthracis attach to
the surface of macrophages, they may be rapidly phagocytized. There can be a
tight interaction
between the exosporium and the phagolysosomal membrane; however, newly
vegetative bacilli
may escape from the phagosomes of cultured macrophages and replicate within
the cytoplasm of
the cells. Release of bacteria from the macrophage occurs 4-6 hours after
phagocytosis of the
spores. The principal virulence factors of B. antlzracis are encoded on
plasmids. One plasmid
(pXO1) carries the toxin genes while a second plasmid (pXO2) encodes the
polyglutamic acid
capsule biosynthetic apparatus.
In certain embodiments, the methods and compositions of the present invention
may also
be used to develop vaccines for other anthrax-like bacteria or microorganisms
of interest. Spores
of anthrax-like infections are similar to those of B. ant/Zracis spores. For
exainple, Bacillus
cereus has been shown to have an exosporiuin that contains glycoproteins,
oligosaccharides, and
other sugars. Also, the B. cereus G9241 vegetative cell can resemble an
anthrax vegatative cell
because both contain a capsule, although the B. cereus G9241 capsule is not
coded for the pXO2
plasmid of B. ant/Zracis, but appears to be encoded for by a pBC218 cluster
(Hoffinaster et al.,
2004). Several of the anthrax toxins encoded for on the pXO1 plasmid may have
similar
counterparts in B. cereus G9241 encoded for on pBC218 including AtxA (toxin
regulator), lethal
factor, and protective antigen (PA). There is evidence that the PA found in B.
cereus G9241 may
be functional, because 27 out of 33 amino acids important to the functionality
of the PA are
identical in B. antlaracis Ames strain and B. cereus G924 1.
Antibodies reactive with the surface of spores of B. antlzracis spores may
affect the
interactions of the spore with host cells and/or the enviroluizent. For
exainple, spore surface
reactive antibodies may enhance phagocytosis of the spores by niurine
peritoneal macrophages,
and may inhibit spore germination in vitro. The =first spore-surface protein,
termed Bc1A
(Bacillus, collagen-like protein) has been recently described in B. anthracis
. The poly-D-
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glutamic acid capsule is not present in the spore, thus surface proteins,
including Bc1A, constitute
the surface layer. Mass spectrometry has been utilized to look for other spore-
specific
constituents of B. anthracis.
The spore is characterized by the presence of 3-0-methyl rhamnose, rhamnose
and
galactosamine . This carbohydrate is found only in the spores and is not
synthesized by
vegetatively growing cells. B. thuYingiensis and B. cereus are closely related
genetically to B.
anthracis and the exosporium of botll contain a glycoprotein whose major
carbohydrate
constituent is rhamnose, while the B. thuringiensis protein additionally
contains galactosamine.
Another sugar monomer is present in the B. tlzuringienisis exosporium, which
can be 3-0-methyl
rhamnose or 2-0-methyl rhamnose, identified previously as spore sugars.
1. Preparation of Compositions
In an embodiment, glycoproteins on the exosporium of the B. anthracis spore
may be
complexed to other proteins, glycoproteins, oligosaccharides, lipids, or
phospholipids. A
diagrammatic representation of a B. antlaf acis bacterium (or other
microorganisms) 2 surround by
a exosporium 4 is provided in FIG. 1. Thus, it can be seen that the spore may
coniprise a variety
of glycoproteins or lippopolysaccharides 5, complexed with other biomolecules
such as sugars or
oligosaccharides 6, peptides 8, lipids 12 and the like. Also, in an
embodiment, at least some of
these coniplexes 14, 16 are antigenic, such that isolation of the antigenic
epitopes may be used to
create an anti-anthrax vaccine. Thus, as disccussed herein, it has been found
that vaccines
comprising only toxin proteins 7,9 (e.g., PA; LF) isolated from the actual
bacterium are not
completely effective against inhalation anthrax. By adding spore-based
antigens to a vaccine,
embodiments of the compositidns of the present invention can provide iinproved
immunity to
anthrax and antlirax-based diseases (or to other disease of interest).
FIG. 2 provides a schematic representation of a method of the present
invention. The
method may comprise two parts which may be performed individually, or in
combination as
shown in FIG. 2. As shown in FIG. 2, in an embodiment, the present invention
provides a
method for purifying glycoproteins and other molecules from the B. anthracis
spore. In an
embodiment, the method may comprise a first step of isolating spores from B.
antlaracis, or
another anthrax-lilce bacterium (or microorganism of interest) 22. Isolation
of the spores may be
performed centrifugation as described in Example 11 herein or other metliods
lcnown in the art
such as high performance liquid chromatography (HPLC). An example of isolated
B. anthracis
spores as isolated by 2D-gel electrophoresis is shown in FIG. 4 (arrows point
to the white
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spores). Next the method may comprise lysing the spores using urea,
sonication, bead beatting,
French press, or some other means 24. Lysing the spores may be performed by
talcing a pure
(about 95-100% purity) spore solution (B. antlaracis spores plus PBS or water)
and performing a
urea extract or some other lysis procedure such as sonicating herein or using
methods known in
the art.
At this point an optional step of purifying complexes from the spores by size-
exclusion
chromatograplly or HPLC 26 may be performed.
Next, the lysed spores, or size-selected fraction may be applied to a column
to purify
glycoproteins contained in the complexes. In an embodiment, , lectin is used
to purify
glycoprotein coinplexes from the spore mixture 28. Lectins are sugar binding
proteins that can
recognize and bind to the carbohydrate portion of a glycoprotein. The lectin
can then be released
from the glycoprotein by washing the lectin with another sugar that has a
stronger affinity for the
lectin than the B. anthracis glycoprotein 30. An example showing a subset of
B. anthracis
proteins purified by lectin-binding is shown in FIG. 3. Thus, it can be seen
that upon extraction
with lectin, a subset of the proteins (e.g., EAl, and new proteins 1, 2, 3, 4,
5, 6, and 7) seen in the
urea extracted spore are isolated. At this point, the eluted glycoprotein may
be identified by time
of flight mass spectrometry (MS-TOF), protein sequencing or other similar
methods 32. For
example, FIG. 5 shows results for MALDI TOF MS of the EAl band seen on the gel
of FIG. 3.
As described herein, the glycoprotein complexes can be used as a vaccine for
immunity against
antlirax infection or any anthrax lilce diseases or as a diagnostic tool for
detection of Bacillus
antlu acis, any other anthrax like spores or where another microorganism of
interest.
In an embodiment, electroelution may be used to delete specific proteins from
the lectin-
purified complexes. Alternatively, electroelution of urea extracted or other
lysed spores may be
used to add proteins to the lectin complexed mixture 34 (FIG. 2). For
electroelution, one or two
dimensional SDS (sodium dodecyl sulfate) PAGE (polyacrylamide gel
electrophoresis) or native
gel electrophoresis of the isolated spore proteins may be performed. The gel
may then be stained,
and the spot of interest cut out, and destained. Next, an electrical charge is
ran through the
isolated portion of the gel containing the protein of interest to elute the
protein from the gel.
Other techniques, such as size exclusion chroinatography or HPLC may be used
to remove
proteins, glycoproteins, lipids, phospholipids, or oligosaccharides outside
the molecular weights
of interest. The eluted protein may be captured on a filter, or in a vessel
such as a tube or filter
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tube, and analyzed by MS-TOF, protein sequencing or other similar methods such
s MALDI
TOF-TOF, ESI-IT, MADLIFT-ICR or ESI FT-ICR MS 36.
In an embodiment, only specific glycoproteins isolated from the lectin coluinn
and
correlating with the spots of interest on a one or two dimensional SDS or
native gel are used to
make the compositions of the present invention (e.g., a vaccine) 33, 40 (FIG.
2). Alternatively,
proteins isolated from the spore complex may be added back to the purified
glycoprotein
complex(es) and used to inalce a composition of the present invention. 33, 38,
40 (FIG. 2).
FIG. 3, panels A and B, shows a representation of the type of results that
maybe obtained
upon upon isolating B. anthracis spore proteins by lectin treatment. Thus, in
an einbodiment, the
profile of proteins in the sample may be characterized by one or two-
dimensional (2D) gel
electrophoresis. In an embodiment the samples are separated iri one dimention
on the basis of
charge along a gradient of increasing pH, as in 2D gel electrophoresis an in
the otlier dimension
on the basis of size. It can be seen that the profile of proteins isolated
from the B. anthracis spore
comprises substantially fewer proteins after lectin' treatment (FIG. 3B) than
before lectin
treatment (FIG. 3A).
2. Vaccines
In an embodiment, the compositions of the present invention comprise a
vaccine. Several
basic strategies may be used to make vaccines against viral and bacterial
infections. U.S. Patent
applications disclosing vaccines to antlirax 'and anthrax lilce infections are
20030118591,
2004/0009178, 2004/0009945, 2002/0142002; these patent applications are
incorporated by
reference herein with respect to material related to anthrax vaccines and the
materials used to
malce anthrax vaccines. The anthrax vaccine containing the protective antigen
(PA) component
of the tripartite anthrax toxin (AVA) is not fully protective in animal
studies. Indeed, a conjugate
vaccine, additionally targeting the poly-D-glutamic acid capsule (PGA), which
surrounds and
protects the vegetative cell from killing by complement mediated killing (Rhie
et al., 2003;
Schneerson et al., 2003), has been sought after. However, such a vaccine would
target the
vegetative cell and letlial toxin, but not the initial interaction of the
macrophage with the spore.
The vaccines disclosed herein may be composed of lectin-purified glycoprotein
complexes isolated from B. anthracis spores. In an einbodiment, the vaccines
are used in
conzbination with other components isolated from the anthrax bacteriuin and/or
spore such as
protective antigen or LF antigen. Or capsule components may be included.
Furthermore, the
vaccine may use lectin-purified glycoprotein complexes isolated from the B.
aiatliracis spores in
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whole or in part, including complexes that may contain deglycosylated forms,
fusion proteins, or
missing or deleted subunits of the glycoprotein complex. In an embodiment,
fragments of a B.
antlaracis lectin binding glycoprotein can be combined with PA fragments. For
example,
fragments of a B. anthracis lectin binding glycoprotein complex can be
combined witli PA
fragments. Or, fragments of a B. antlaracis lectin binding glycoprotein
complexes can be
combined with other spore associated antigens such as extractable antigen
1(EA1), Serum
Amyloid P Component (SAP) or capsular poly-gamma-d-glutamic acid (PGA). In
another
embodiment, the present-invention relates to an anthrax vaccine comprising one
or more replicon
particles derived from one or more replicons encoding one or more B.
antlaracis proteins or
polypeptides.
In an embodiment, the vaccines of the present invention comprise an adjuvant
to increase
the humoral and/or cellular immune response. In an embodiment, the adjuvant is
one that is
approved by the Food and Drug Administration such as aluminum hydroxide and
aluminum
phosphate. Or the Ribi adjuvant can be employed.
3. Vaccine Administration
The peptides, compositions, vaccines or antibodies disclosed herein can be
administered
by any mode of administration capable of delivering a desired dosage to a
desired location for a
desired biological effect which are known to those of ordinary skill in the
art. Routes or modes
include, for example, oral administration, parenteral administration (e.g.,
intravenously, 'by
intramuscular injection, by intraperitoneal injection), or by subcutaneous
administration. In an
embodiment, the vaccine is prepared for subcutaneous or intramuscular
injection. The vaccine
may be formulated in such a way as to render it deliverable to a mucosal
membrane without the
peptides being broken down before providing systemic or mucosal immunity, such
as, orally,
inhalationally, intranasally, or rectally. The amount of active compound
administered will, of
course, be dependent, for example, on the subject being treated, the subject's
weight, the manner
of adininistration and the judgment of the prescribing physician. Immunogenic
amounts can be
determined by standard procedures. An "immunogenic aniount" is an amount of
the protein
sufficient to evoke an immune response in the subject to which the vaccine is
administered. An
amount of from about 102 to 10 7 microgranis per lcilogram dose is suitable,
with more or less
used depending upon the age and species of the subject being treated.
Depending on the intended mode of administration, the coinpositions or
vaccines may be
in the form of solid, semi solid or liquid dosage forms, sucli as, for
example, tablets,
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suppositories, pills, capsules, powders, liquids, suspensions, or the like,
preferably in unit dosage
form suitable for single administration of a precise dosage. The compositions
or vaccines may
include, as noted above, an effective amount of the selected iminunogens in
combination with a
pharmaceutically acceptable carrier and, in addition, may include other
medicinal agents,
pharmaceutical agents, carriers, adjuvants, diluents, etc. Exemplary
pharmaceutical carriers
include sterile pyrogen-free water and sterile pyrogen-free physiological
saline solution.
Parental administration can involve the use of.a slow release or sustained
release system,
such that a constant level of dosage is maintained. See, e.g., U.S. Patent No.
3,710,795, which is
incorporated by reference herein. A system using slow release or sustained
release may be used
with oral administration as well. The vaccine or composition can be
administered in liposomes,
encapsulated, or otherwise protected or formulated for slower or sustained
release. The antibody
level following the first exposure to a vaccine antigen referred to as primary
antibody response
may consist primarily of IgM, and may be of brief duration and low intensity,
so as to be
inadequate for effective protection. The antibody level following the second
and subsequent
antigenic challenges, or secondary antibody response, may appear more quiclcly
and persists for a
longer period, attain a higher titer, and consists predominantly of IgG. The
shorter latent period
is generally due to antigen-sensitive cells, called memory cells, already
present at the time of
repeat exposure.
In an embodiment, the vaccine is provided as an adenovirus vector. In an
embodiment,
the adenovirus-based vaccine can be administrated by different routes to
achieve immunization
such as intramuscular injection (parentally), intranasal administration or
oral administration. The
intranasal immunization with this type of vaccine may be preferred to elicit
more potent mucosal
immunity against the pathogen, in this case, anthrax spores. In an embodiment,
intranasal
administration may be provided for protection against inhalation anthrax
caused by aerosol
dismissed anthrax spore propagated by a bioterrorism attack.
Anthrax vaccines as currently administered can function with six immunizations
over a
period of 18 months followed by annual boosters. In an enlbodiment, the
vaccines of the present
invention may be provided with 1, 2, 3, 4, or 5 iinmunizations to provide
protective immunity
with optional boosters. Examples of suitable iminunization schedules include,
but are not limited
to: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1
inontli, (iv) 0 and 6
months, or other schedules sufficient to elicit the desired inimune responses
expected to confer
protective immunity, or reduce disease symptoms, or reduce severity of
disease.
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In an embodiment, the vaccine of the present invention may provide at least
one of anti-
glycoprotein complex IgG antibody titers, anti-glycoprotein complex IgGl
antibody titers, anti-
glycoprotein complex IgG2a antibody titers. In alternate embodiments, antibody
titers of 100,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500,
4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000,
11500, and 12000
by 2, 4, 6, 8; 10, 12, 14, 16, 18, and 20 weeks post-immunization following 1,
2, 3, 4, 5, or more
immunizations are achieved. In an einbodiment, booster inoculations are used
to maintain
effective immunization. Boosters can be given every 1, 2, 3, 4, 6, 8, 12 years
following prior
inoculation, for example.
In an embodiment, the vaccine may comprise a nucleic acid that encode for an
immunogenic anthrax protein or polypeptide isolated by the methods of the
present invention.
For example, in an embodiment, a nucleic acid comprising a nucleic acid
sequence included in
the sequences as set forth in SEQ ID NOs: 1-26 may be used in a vaccine of the
present
invention.
When DNA (or RNA corresponding to the DNA sequence) is used as a vaccine, the
DNA
(or RNA) can be administered directly using techniques such as delivery on
gold beads (gene
gun), delivery by liposomes, or direct injection, anzong other methods known
to people in the art.
Any one or more constructs or DNA or RNA can be use in any combination
effective to elicit an
immunogenic response in a subject. Generally, the nucleic acid vaccine
adininistered may be in
an amount of about 1-5 g of nucleic acid per dose and will depend on the
subject to be treated,
capacity of the subj ect's immune system to develop the desired immune
response, and the degree
of protection desired. Precise amounts of the vaccine to be administered may
depend on the
judgment of the practitioner and may be peculiar to each subject and antigen.
4. Assays for Assessing the Immune Response
Embodiments of the present invention also provide assays for assessing an
immune
response to the components isolated from the endosporium of B. anthracis.
The assays may comprise in vivo assays, such as assays to measure antibody
responses
and delayed type hypersensitivity responses. In an embodiment, the assay to
measure antibody
responses primarily may measure B-cell function as well as B-cell/T-cell
interactions. In another
embodiment, the delayed type hypersensitivity response assay may measure T-
cell immunity.
For the antibody response assay, antibody titers in the blood may be conipared
following an
antigenic challenge. These levels can be quantitated according to the type of
antibody, as for
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example, IgG, IgGl, IgG2, IgM, or IgD. Also, the development of immune systems
may be
assessed by determining levels of antibodies and lymphocytes in the blood
without antigenic
stimulation. An agglutination assay to test the highest dilution of antibodies
that can still bind to
B. anthracis spores or any other strain of anthrax may be used.
The assays may also comprise in vitro assays. The in vitro assays may comprise
determining the ability of cells to divide, or to provide help for other cells
to divide, or to release
lyinophokines and other factors, express marlcers of activation, and lyse
target cells. Lymphocytes
in mice and man can be compared in vitro assays. In an embodiment, the
lymphocytes from
similar sources such as peripheral blood cells, spleenocytes, or lymphnode
cells, are compared. It
is possible, however, to compare lymphocytes from different sources as in the
non-limiting
example of peripheral blood cells in humans and splenocytes in mice. For the
in vitro assay, cells
may be purified (e.g., B-cells, T-cells, and macrophages) or left in their
natural state (e.g.,
splenocytes or lymph node cells). Purification may be by any method that gives
the desired
results. The cells can be tested in vitro for their ability to proliferate
using mitogens or specific
antigens. Mitogens can specifically test the ability of-either T-cells to
divide as in the non-
limiting examples of concanavalin A and T-cell receptor antibodies, or B-cells
to divide as in the
non-limiting example of phytohemagglutinin. The ability of cells to divide in
the presence of
specific antigens can be determined using a mixed lymphocyte reaction, MLR,
assay. Supernatant
from the cultured cells can be tested to quantitate the ability of the cells
to secrete specific
lymphokines. The cells can be removed from culture and tested for their
ability to express
activation antigens. This cai be done by any method that is suitable as in the
non-limiting
example of using antibodies or ligands to which bind the activation antigen as
wellas probes that
bind the RNA coding for the activation antigen.
Also, in an embodiment, phenotypic cell assays can be performed to determine
the
frequency of certain cell types. Peripheral blood cell counts may be performed
to determine the
number of lymphocytes or macrophages in the blood. Antibodies can be used to
screen peripheral
blood lymphocytes to determine the percent of cells expressing a certain
antigen as in the non-
limiting example of determining CD4 cell counts and CD4/CD8 ratios.
In certain embodiments, transformed host cells can be used to analyze the
effectiveness of
drugs and agents which inhibit anthrax or B. aratlaracis proteins, such as
host proteins or
chemically derived agents or other proteins which may interact with B.
aratlaracis proteins of the
present invention to inhibit its fiunction. A method for testing the
effectiveness of an anti-anthrax
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drug or anti-anthrax like diseases drug or agent can for example be the rat
anthrax toxin assay
(Ivins et al. 1986, Infec. Immun. 52, 454-458; and Ezzell et al., Infect.
Immun., 1984, 45:761-
767) or a skin test in rabbits for assaying antiserum against anthrax toxin
(Belton and Henderson,
1956, Br. J. Exp. Path. 37, 156-160).
5. Generation of Antibodies
Other embodiments of the present invention comprise generation of antibodies
that
specifically recognize a lectin-binding glycoprotein isolated from the
endosporium of the B.
anthracis spore alone, or in combination with other B. anthracis components.
In an embodiment,
the antibody preparation, whether polyclonal, monoclonal, chimeric, human,
huinanized, or non-
human can recognize and target the variants and fragments a lectin-binding
glycoprotein complex
isolated from the. B. antlzracis spore alone, or in combination with other B.
anthracis
components. Antibodies that specifically recognize non-native variants or
fraginents of any of the
lectin-binding glycoprotein complexes isolated from the endosporium of the B.
anthracis spore
alone, or in combination with other B. anth>~acis components could, for
example, be used to
purify recombinant fragments lectin-binding glycoprotein complexes isolated
from the
endosporium of the B. anthracis spore and variants of such proteins. Such
antibodies could also
be used as "passive vaccines" for the direct immunotherapeutic targeting
ofBacillus anthracis in
vivo. Also disclosed are methods of using said antibodies to detect anthrax
spores or spore
fragments, either in vitro or in vivo, for research or diagnostic use.
In an embodiment, the antibodies provided herein are capable of neutralizing
anthrax
spores and spores of other closely related species to anthrax. The provided
antibodies can be
delivered directly, such as through needle injection, for example, to treat
anthrax or anthrax-like
infections. The provided antibodies can be delivered non-invasively, such as
intranasally, to treat
inhalation anthrax or anthrax-like diseases.
In an embodiment, the antibodies may be encapsulated, for example into
lipsomes,
microspheres, or other transfection enhancement agents, for improved delivery
into the cells to
maximize the treatment efficiency. In an embodiment, the DNA sequences
encoding the
provided antibodies, or their fragments such as Fab fraginents, may be cloned
into genetic
vectors, such as plasmid or viral vectors, and delivered into the hosts for
endogenous expression
of the antibodies for treatment of anthrax or anthrax-like diseases.
In an embodiment, the antibodies are generated in other species and
"humanized" for
administration in humans. Humanized forms of non-human (e.g., murine)
antibodies are
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chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as
Fv, Fab, Fab',
F(ab')2, or other antigen-binding subsequences of antibodies) which contain
minimal sequence
derived from non-human iminunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a complementary
determining
region (CDR) of the recipient are replaced by residues from a CDR of a non-
human species
(donor antibody) such as mouse, rat or rabbit having the desired specificity,
affinity and capacity.
In some instances, Fv framework residues of the human immunoglobulin are
replaced by
corresponding non-human residues. Humanized antibodies may also comprise
residues that are
found neither in the recipient antibody nor in the imported CDR or frameworlc
sequences. In
general, the humanized antibody will comprise substantially all of at least
one, and typically two,
variable domains, in which all or substantially all of the CDR regions
correspond to those of a
non-human iinmunoglobulin and all or substantially all of the FR regions are
those of a human
immunoglobulin consensus sequence. The lluinanized antibody optimally also
will comprise at
least a portion of an immunoglobulin constant region (Fc), typically that of a
liuman
immunoglobulin (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,
1988, Nature,
332:323-327; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596 .
Methods for humanizing non-human antibodies known in the art may be used to
humanize the antibodies of the present invention. Generally, a htunanized
antibody has one or
more amino acid residues introduced into it from a source that is non-human.
These non-human
amino acid residues are often referred to as "import" residues, which are
typically talcen from an
"import" variable domain. Humanization can be essentially performed by
substituting rodent
CDRs or CDR sequences for the corresponding sequences of a human antibody (see
e.g., Jones et
al., 1986, Nature, 321:522-525; Riechmann et a1.,1988, Nature, 332:323-327;
Verhoeyen et al.,
1988, Science, 239:1534-1536. Accordingly, such "humanized" antibodies are
chimeric
antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an
intact htiman variable
domain has been substituted by the corresponding sequence from a non-human
species. In
practice, humanized antibodies are typically human antibodies in wliich some
CDR residues and
possibly some FR residues are substituted by residues from analogous sites in
rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in
making the
humanized antibodies may be highly important in order to reduce antigenicity.
According to the
"best-fit" method, the sequence of the variable domain of a rodent antibody is
screened against
the entire library of known human variable domain sequences. The liuman
sequence which is
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closest to that of the rodent is then accepted as the human frameworlc (FR)
for the humanized
antibody (Sims et al., 1993, J. Immunol., 151:2296; Chothia et al., 1987, J.
Mol. Biol., 196:901.
Another method uses a particular frameworlc derived from the consensus
sequence of all human
antibodies of a particular subgroup of light or heavy chains. The same
frainework may be used
for several different humanized antibodies (Carter et al., Proc. Natl. Acad.
Sci. USA, 1992,
89:4285; Presta et al., J. Immunol., 1993, 151:2623).
In an embodiment, the antibodies are humanized with retention of high affinity
for the
antigen and other favorable biological properties. To achieve this goal the
humanized antibodies
may be prepared by analysis of the parental sequences and various conceptual
humanized
products using three dimensional models of the parental and humanized
sequences.
Computerized comparison of these displays to publicly available three
dimensional
immunoglobulin models permits analysis of the likely role of the residues in
the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of
the candidate inimunoglobulin to bind its antigen. In this way, the human
framework (FR)
residues can be selected and combined from the consensus and import sequence
so that the
desired antibody characteristic, such as increased affinity for the target
antigen(s), is acliieved. In
general, the CDR residues are directly and most substantially involved in
influencing antigen
binding (see e.g., WO 94/04679).
In an embodiment, transgenic animals (e.g., mice) that are capable, upon
immunization,
of producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin
production can be employed. For example, it has been described that the
liomozygous deletion of
the antibody heavy chain joining region JH gene in chimeric and germ-line
mutant mice results in
complete inhibition of endogenous antibody production. Transfer of the human
germ-line
immunoglobulin gene array in such genn-line mutant inice can result in the
production of human
antibodies upon antigen challenge (see, e.g., Jalcobovits et al., 1993, Proc.
Natl. Acad. Sci. USA,
90:2551-2555; Jalcobovits et al., 1993, Nature, 362:255-258; Bruggemann et
al., 1993, Year in
Immunology, 7:33).
In yet another embodiment, human antibodies may also be produced in phage
display
libraries (Hoogenboom et al., 1991, J. Mol. Biol., 227:381; Marlcs et al.,
1991, J. Mol. Biol.,
222:581. In another embodiment, the antibodies are monoclonal antibodies (see
e.g., Cole et al.,
1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner
et al., 1991, J.
Immunol., 147(1):86-95. For example, the present invention may coinprise
hybidoma cells that
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produce monoclonal antibodies. Monoclonal antibodies may be prepared using
liybridoma
methods (see e.g., Kohler and Milstein, 1975, Nature, 256:495; or Harlow and
Lane, 1988,
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New Yorlc).
In a hybridoma
method, a mouse or other appropriate host animal, is typically iminunized with
an immunizing
agent to elicit lymphocytes that produce or are capable of producing
antibodies that will
specifically bind to the immunizing agent. Alternatively, the lymphocytes may
be immunized in
vitro. Preferably, the immunizing agent comprises a composition comprising at
least one
glycoprotein on the exosporium of the Bacillus anthracis spore where the
glycoprotein comprises
at least one lectin-binding sugar. Traditionally, the generation ofmonoclonal
antibodies has
depended on the availability of purified protein or peptides for use as the
immunogen. More
recently DNA based immunizations have shown promise as a way to elicit strong
iminune
responses and generate monoclonal antibodies. In this approach, DNA-based
immunization can
be used, wherein DNA encoding a portion of the anthrax spores expressed as a
fusion protein
with human IgGI is injected into the host animal according to methods known in
the art (e.g.,
Kilpatrick KE, et al., 1998, Hybridoma, Dec. 17(6):569-76; Kilpatrick KE et
al., 2000,
Hybridoma, Aug., 19(4):297-302) and as described in the exainples.
In yet another embodiment, the antigen may be expressed in baculovirus. The
advantages
to the baculovirus system include ease of generation, high levels of
expression, and post-
translational modifications that are highly siniilar to those seen in
mainmalian systems. The
antigen is produced by inserting a gene encoding the B. anthracis antigenic
protein so as to be
operably linked to a signal sequence such that the antigen is displayed on the
surface of the
virion. This method allows immunization with whole virus, eliminating the need
for purification
of target antigens.
In an embodiment, peripheral blood lyinphocytes ("PBLs") are used in methods
of
producing monoclonal antibodies if cells of human origin are desired. In an
alternate
embodiment, spleen cells or lymph node cells may be used if non-human
mammalian sources are
desired. The lymphocytes are then fiised with an immortalized cell line using
a suitable fiising
agent, such as polyethylene glycol, to form a hybridoma cell (Goding,
"Monoclonal Antibodies:
Principles and Practice" Academic Press, (1986) pp. 59-103). Iinmortalized
cell lines may be
transformed mammalian cells, including myeloma cells of rodent, bovine,
equine, and lluman
origin. In an embodiment, rat or mouse niyeloma cell lines, are employed. The
hybridoma cells
may be cultured in a suitable culture medium that preferably contains one or
more substances that
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inhibit the growth or survival of the unfused, immortalized cells. For
example, if the parental
cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT
or HPRT), the
culture medium for the hybridomas typically will =include hypoxanthine,
aminopterin, and
thymidine ("HAT medium"), which substances prevent the growth of HGPRT-
deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level
expression of antibody by the selected antibody-producing cells, and are
sensitive to a medium
such as HAT medium. More preferred immortalized cell lines are murine myeloma
lines, which
can be obtained, for instance, from the Salk Institute Cell Distribution
Center, San Diego, Calif.
and the American Type Culture Collection, Rockville, Md. Human myeloma and
inouse-human
heteromyeloma cell lines also have been described for the production of human
monoclonal
antibodies (Kozbor, 1984, J. Immunol., 133:3001; Brodeur et al., 1987,
"Monoclonal Antibody
Production Techniques and Applications" Marcel Deklcer, Inc., New York, pp. 51-
63). The
culture medium in which the hybridoma cells are cultured can then be assayed
for the presence of
monoclonal antibodies directed against the B. antlzracis antigen.
In an embodiment, the binding specificity of monoclonal antibodies produced by
the
hybridoma cells may be determined by immunoprecipitation or by an in vitro
binding assay, such
as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques
and assays are known in the art, and are described further in the Examples
below or in Harlow
and Lane "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications,
New York,
(1988).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting
dilution or FACS sorting procedures and grown by standard methods. Suitable
culture media for
this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-
1640
medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in
a mammal. The
monoclonal antibodies secreted by the subclones may be isolated or purified
from the culture
medium or ascites fluid by conventional immunoglobulin purification procedures
such as, for
example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel
electrophoresis,
dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recoinbinant DNA methods, such
as
those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal
antibodies can be
readily isolated and sequenced using conventional procedures (e.g., by using
oligonucleotide
probes that are capable of binding specifically to genes encoding the heavy
and light chains of
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murine antibodies). The hybridoma cells serve as a preferred source of such
DNA. Once
isolated, the DNA may be placed into expression vectors, which are then
transfected into host
cells such as simian COS cells, Chinese hamster ovary (CHO) cells,
plasmacytoma cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to obtain
the synthesis of
monoclonal antibodies in the recombinant host cells. The DNA also may be
modified, for
example, by substituting the coding sequence for hui-nan heavy and light chain
constant domains
in place of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by
covalently joining
to the immunoglobulin coding sequence all or part of the coding sequence for a
non-
imniunoglobulin polypeptide. Optionally, such a non-immunoglobulin polypeptide
is substituted
for the constant domains of an antibody or substituted for the variable
domains of one antigen-
combining site of an antibody to create a chimeric bivalent antibody
comprising one antigen-
combining site having specificity for anthrax spores and anthrax-like other
species.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of
antibodies to produce fragments thereof, particularly, Fab fragments, can be
accomplished using
routine techniques known in the art. For instance, digestion can be perfonned
using papain.
Examples of papain digestion are described in WO 94/29348; U.S. Pat. No.
4,342,566; and
Harlow and Lane, Antibodies, 1988, A Laboratory Manual, Cold Spring Harbor
Publications,
New Yorlc. Papain digestion of antibodies typically produces two identical
antigen binding
fragments, called Fab fragments, each with a single antigen binding site, and
a residual Fc
fragment. Pepsin treatment yields a fragment, called the F(ab')2 fragment,
that has two antigen
combining sites and is still capable of cross-linking antigen. The Fab
fragments produced in the
antibody digestion also contain the constant domains of the light chain and
the first constant
domain of the heavy chain. Fab' fragments differ from Fab fiagments by the
addition of a few
residues at the carboxy terminus of the heavy chain domain including one or
more cysteines from
the antibody hinge region. The F(ab')2 fraginent is a bivalent fragment
comprising two Fab'
fragments linked by a disulfide bridge at the hinge region. Fab'-SH is the
designation herein for
Fab' in which the cysteine residue(s) of the constant domains bear a free
thiol group. Antibody
fragments originally were produced as pairs of Fab' fragments which have hinge
cysteines
between them. Other chemical couplings of antibody fragments are also lcnown.
In other embodiments, an isolated immunogenically specific paratope or
fraginent of the
antibody is also provided. A specific iinmunogenic epitope of the antibody can
be isolated from
the whole antibody by chemical or rnechanical disruption of the molecule. The
purified
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fragments thus obtained may then be tested to detennine their iminunogenicity
and specificity by
the methods described herein. Irnmunoreactive paratopes of the antibody,
optionally, are
synthesized directly. An immunoreactive fragment is defined as an amino acid
sequence of at
least about two to five consecutive amino acids derived from the antibody
amino acid sequence.
In another embodiment, the antibodies of the present invention may be made by
linking
two or more peptides or polypeptides togetller by protein chemistry
techniques. For example,
peptides or polypeptides can be chemically synthesized using currently
available laboratory
equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -
butyloxycarbonoyl)
chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art
can readily
appreciate that a peptide or polypeptide corresponding to the antibody, for
example, can be
synthesized by standard chemical reactions. For exainple, a peptide or
polypeptide can be
synthesized and not cleaved from its synthesis resin whereas the other
fragment of an antibody
can be synthesized and subsequently cleaved from the resin, thereby exposing a
terminal group
which is functionally blocked on the other fragment. By peptide condensation
reactions, these
two fraginents can be covalently joined via a peptide bond at their carboxyl
and amino termini,
respectively, to form an antibody, or fragment thereof. (Grant GA (1992)
Synthetic Peptides: A
User Guide. W.H. Freeman and Co., N.Y. (1992); Bodanslcy M and Trost B., Ed.
(1993)
Principles of Peptide Synthesis. Springer-Verlag Inc., NY. Alternatively, the
peptide or
polypeptide may be independently synthesized iia vivo as described above. Once
isolated, these
independent peptides or polypeptides may be linked to form an antibody or
fragment thereof via
similar peptide condensation reactions.
For exaniple, in an embodiment, enzymatic ligation of cloned or synthetic
peptide
segments allow relatively short peptide fragments' to be joined to produce
larger peptide
fraginents, polypeptides or whole protein domains (Abrahmsen L et al.,
Biochemistry, 30:4151
(1991)). Alternatively, native chemical ligation of synthetic peptides can be
utilized to
synthetically construct large peptides or polypeptides from shorter peptide
fragments. This
method consists of a two step chemical reaction (Dawson et al., 1994, Science,
266:776-779).
The first step is the chemoselective reaction of an unprotected syntlietic
peptide-alpha-thioester
with another unprotected peptide segment containing an amino-terminal Cys
residue to give a
thioester-linked interinediate as the initial covalent product. Without a
change in the reaction
conditions, this intennediate undergoes spontaneous, rapid intramolecular
reaction to form a
native peptide bond at the ligation site. Application of this native chemical
ligation method to
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the total synthesis of a protein molecule is illustrated by the preparation of
liuman interleulcin 8
(IL-8) (Baggiolini M et al., 1992, FEBS Lett. 307:97-101; Clarlc-Lewis I et
al.,1994,
J.Biol.Chem., 269:16075); Clark-Lewis I. et al.,1991, Biochemistry, 30:3128;
Rajarathnam K et
al., 1994, Biochemistry 33:6623-30).
Alternatively, unprotected peptide segments may be chemically linked where the
bond
formed between the peptide segments as a result of the chemical ligation is an
umiatural (non-
peptide) bond (Schnolzer, M et al., 1992, Science, 256:221). This technique
has been used to
synthesize analogs of protein domains as well as large amounts of relatively
pure proteins with
full biological activity (deLisle Milton RC et al., 1992, Techniques in
Protein Chemistry IV.
Academic Press, New Yorlc, pp. 257-267).
Also disclosed are fraginents of antibodies which have bioactivity. The
polypeptide
fragments can be recombinant proteins obtained by cloning nucleic acids
encoding a
glycoprotein of the B. anthracis spore polypeptide in an expression system
capable of producing
the polypeptide fragments thereof, such as an adenovirus or baculovinis
expression system. For
example, one can determine the active domain of an antibody from a specific
hybridoma that can
cause a biological effect associated with the interaction of the antibody with
anthrax spores or
spores of other closely related species. Amino acids found to not contribute
to either the activity
or the binding specificity or affinity of the antibody can be deleted without
a loss in the respective
activity. For example, in various embodiments, amino or carboxy-terminal amino
acids are
sequentially removed from either the native or the modified non-immunoglobulin
molecule, or
the immunoglobulin molecule, and the respective activity assayed in one of
many available
assays. In another example, a fragment of an antibody comprises a modified
antibody wllerein at
least one amino acid has been substituted for the naturally occurring amino
acid at a specific
position, and a portion of either amino terminal or carboxy temlinal anlino
acids, or even an
internal region of the antibody, has been replaced with a polypeptide fragment
or other moiety,
such as biotin, which can facilitate in the purification of the modified
antibody. For example, a
modified antibody can be fused to a maltose binding protein, through either
peptide chemistry or
cloning the respective nucleic acids encoding the two polypeptide fraginents
into an expression
vector such that the expression of the coding region results in a hybrid
polypeptide. The hybrid
polypeptide can be affinity purified by passing it over an ainylose affinity
column, and the
modified antibody receptor can then be separated from the maltose binding
region by cleaving the
hybrid polypeptide with the specific protease factor Xa. (See, for example,
New England Biolabs
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WO 2007/044607 PCT/US2006/039293
Product Catalog, 1996, pg. 164.). Similar purification procedures are
available for isolating
hybrid proteins from eulcaryotic cells as well.
The fragment of the B. anthracis spore polypeptide, whether attached to other
sequences
or not, include insertions, deletions, substitutions, or other selected
modifications of particular
regions or specific amino acids residues, provided the activity of the
fragment is not significantly
altered or impaired compared to the non-modified antibody or antibody
fragment. These
modifications can provide for some additional property, such as to remove or
add amino acids
capable of disulfide bonding, to increase its bio-longevity, to alter its
secretory characteristics,
etc. In any case, the fragment inust possess a bioactive property, such as
binding activity,
regulation of binding at the binding domain, etc. Functional or active regions
of the antibody
may be identified by mutagenesis of a specific region of the protein, followed
by expression and
testing of the expressed polypeptide. Such methods are readily apparent to a
slcilled practitioner
in the art and can include site-specific mutagenesis of the nucleic acid
encoding the antigen.
(Zoller MJ et al., 1982, Nucl. Acids Res. 10:6487-500). A variety of
iinmunoassay formats may
be used to select antibodies that selectively bind with a particular protein,
variant, or fragment.
For example, solid-phase ELISA immunoassays are routinely used to select
antibodies selectively
immunoreactive with a protein, protein variant, or fragment thereof (Harlow
and Lane, 1988).
In yet another embodiment, the present invention comprises an antibody reagent
kit
comprising containers of the monoclonal antibody to at least one of the sugar
complexed
components of the Bacillus arzthracis spore where the complex comprises at
least one lectin-
binding sugar or fragment thereof and one or more reagents for detecting
binding of the antibody
or fragment tliereof to at least one of the sugar complexed conzponents on the
Bacillus ayathracis
spore where the glycoprotein comprises at least one lectin-binding sugar. The
reagents can
include, for example, fluorescent tags, enzymatic tags, or other tags. The
reagents can also
include secondary or tertiary antibodies or reagents for enzymatic reactions,
wherein the
enzymatic reactions produce a product that can be visualized.
6. Functional Nucleic Acids .
In an embodiment, the compositions of the present invention comprise a
functional
nucleic acid as a therapeutic agent for the treatment or prevention of
anthrax, anthrax-like
infections or otlier diseases of interest. Functional nucleic acids are
nucleic acid molecules that
have a specific function, such as binding a target molecule or catalyzing a
specific reaction. For
exainple, functional nucleic acids include antisense molecules, aptainers,
ribozynies, triplex
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forming molecules, and external guide sequences. The functional nucleic acid
molecules can act
as affectors, inhibitors, modulators, and stimulators of a specific activity
possessed by a target
molecule, or the functional nucleic acid molecules can possess a de novo
activity independent of
any other molecules.
Functional nucleic acid molecules can interact with any macromolecule, such as
DNA,
RNA, polypeptides, or carbohydrate chains. In an embodiinent, the functional
nucleic acid of the
present invention can interact with the mRNA encoding for at least one
glycoprotein on the
exosporium of the Bacillus anthracis spore where the glycoprotein coinprises
at least one lectin-
binding sugar. In yet anotlier embodiment the functional nucleic acid of the
present invention
can interact witli at least one glycoprotein on the exosporium of the Bacillus
anthracis spore
where the glycoprotein comprises at least one lectin-binding sugar. Or, the
functional nucleic
acid of the present invention may interact with the genomic DNA encoding for
at least one
glycoprotein on the exosporium of the Bacillus anthracis spore where the
glycoprotein comprises
at least one lectin-binding sugar. The functional nucleic acids may be
designed to interact with
other B. aratlaracis nucleic acids based on sequence homology between the
target molecule and
the functional nucleic acid molecule. In other embodiments, the specific
recognition between
the functional nucleic acid molecule and the target molecule is not based on
sequence homology
between the functional nucleic acid molecule and the target molecule, but
rather is based on the
formation of tertiary structure that allows specific recognition to talce
place.
In an einbodiment, the functional nucleic acid may comprise an antisense
nucleic acid.
Antisense molecules are designed to interact with a target nucleic acid
molecule through either
canonical or non-canonical base pairing. The interaction of the antisense
molecule and the target
molecule is designed to promote the destruction of the target molecule
tluougli, for exanlple,
RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense
molecule may be
designed to interrupt a processing function that normally would talee place on
the target
molecule, such as transcription or replication. Antisense molecules can be
designed based on the
sequence of the target molecule. Numerous methods for optimization of
antisense efficiency by
finding the most accessible regions of the target molecule exist. Exemplary
metliods may include
in vitro selection experiments and DNA modification studies using DMS and
DEPC. In alternate
embodiments, antisense molecules bind the target molecule with a dissociation
constant (lcd)less
than or equal to 10-6, 10"$, 10-10, or 10"12 M. A representative sainple of
inetllods and techniques
which aid in the design and use of antisense molecules can be found in the
following U.S. patent
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Nos.:'5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607,
5,786,138, 5,849,903,
5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095,
6,007,995,
6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,
6,046,319, and
6,057,437.
In another embodiment, the functional nucleic acid may coinprise an aptamer.
Aptamers
are molecules that interact with a target molecule, preferably in a specific
way. Typically
aptamers are small nucleic acids ranging from 15-50 bases in length that fold
into defined
secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers
can bind small
molecules, such as ATP (U.S. Patent No. 5,631,146) and theophylline (U.S.
Patent No.
5,580,737), as well as large molecules, such as reverse transcriptase (U.S.
Patent No. 5,786,462)
and thrombin (U.S. Patent No.: 5,543,293). In an embodiment, the aptamers of
the present
invention can bind very tightly to the target molecule with a dissociation
constant (lcd) of less than
10"12 M. In alternate embodiments, the aptamers may bind the target molecule
with a lcd less than
10-6, 10"8, 10-10, or 10"12 M. The aptainers of the present invention can bind
the target molecule
witli a very high degree of specificity. For example, aptamers have been
isolated that have
greater than a 10,000 fold difference in binding affinities between the target
molecule and another
molecule that differ at only a single position on the molecule (U.S. Patent
No. 5,543,293). In
alternate embodiments, the aptamer may have a kd with the target molecule at
least 10, 100, 1000,
10,000, or 100,000 fold lower than the lcd with a background binding molecule
such as serum
albumin. Representative exainples of how to malce and use aptainers to bind a
variety of
different target molecules can be found in the following non-limiting list of
United States Patent
Nos: 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613,
5,795,721, 5,846,713,
5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020,
6,013,443,
6,020,130, 6,028,186, 6,030,776, and 6,051,698.
In another embodiment, the composition may comprise a ribozyme. Ribozymes are
nucleic acid molecules that are capable of catalyzing a chemical reaction,
either intramolecularly
or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is
preferred that the ribozynies
catalyze intermolecular reactions. There are a number of different types of
ribozymes that
catalyze nuclease or nucleic acid polymerase type reactions which are based on
ribozymes found
in natural systems, such as hammerhead ribozymes (e.g., U.S. Patent Nos:
5,334,711, 5,436,330,
5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463,
5,861,288,
5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, and
international patent
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WO 2007/044607 PCT/US2006/039293
applications WO 9858058,WO 9858057, and WO 9718312) hairpin ribozymes (e.g.,
U.S. Patent
Nos: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701,
5,869,339, and
6,022,962), and tetrahymena ribozymes (e.g., U.S. PatentNos: 5,595,873 and
5,652,107). There
are also a number of ribozymes that are not found in natural systems, but
which have been
engineered to catalyze specific reactions de novo (e.g., U.S. Patent Nos:
5,580,967, 5,688,670,
5,807,718, and 5,910,408). In ari embodiment, the ribozyme may cleave RNA
substrates.
Ribozymes typically cleave nucleic acid substrates tlirough recognition and
binding of the target
substrate with subsequent cleavage. This recognition is often based inostly on
canonical or non-
canonical base pair interactions. This property makes ribozymes particularly
good candidates for
target specific cleavage of nucleic acids because recognition of the target
substrate is based on the
target substrates sequence. Representative examples of how to make and use
ribozyines to
catalyze a variety of different reactions can be found in the following non-
limiting list of United
States patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855,
5,869,253, 5,877,021,
5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.
In another embodiment, the composition may coinprise a triplex forming nucleic
acid.
Triplex forming functional nucleic acid molecules are molecules that can
interact with either
double-stranded or single-stranded nucleic acid. When triplex molecules
interact with a target
region, a structure called a triplex is formed, in which there are three
strands of DNA forming a
complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex
molecules are
preferred because they can bind target regions with high affinity and
specificity. In alternate
enlbodiments, the triplex forming molecules bind the target molecule with a kd
less than 10"G,10-.
8, 10-10, or 10-12 M. Representative examples of how to malce and use triplex
forming molecules
to bind a variety of different target molecules can be found in the following
non-limiting list of
United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773,
5,834,185,
5,869,246, 5,874,566, and 5,962,426.
In another embodiment, the composition may comprise an external guide
sequences
(EGSs). External guide sequences (EGSs) are molecules that bind a target
nucleic acid molecule
forming a complex, and this complex is recognized by RNase P, which cleaves
the target
molecule. EGSs can be designed to specifically target a RNA molecule of
clioice. RNAse P aids
in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be
recruited to cleave
virtually any RNA sequence by using an EGS that causes the target RNA:EGS
complex to mimic
the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altnian,
Science 238:407-
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WO 2007/044607 PCT/US2006/039293
409 (1990)). Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be
utilized to
cleave desired targets within eukaryotic cells. (Yuan et al., Proc. Natl.
Acad. Sci. USA, 1992,
89:8006-8010; WO 93/22434; WO 95/24489; Yuan and Altman, EMBO J., 1995, 14:159-
168,
and Carrara et al., Proc. Natl. Acad. Sci. (USA), 1995, 92:2627-2631.
Representative examples
of how to make and use EGS molecules to facilitate cleavage of a variety of
different target
molecules be found in the following non-limiting list of United States
patents: 5,168,053,
5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.
7. Peptides
In an embodiment, the composition and/or vaccine of the present invention may
comprise
a polypeptide fragment of at least one glycoprotein on the exosporium of the
Bacillus antlaracis
spore where the glycoprotein comprises at least one lectin-binding sugar. The
peptide can be an
antigen or the antigen bound to a carrier or a mixture of bound or unbound
antigens. The peptide
can then be used in a method of preventing anthrax infection or anthrax-like
infections. For
example, in an embodiment, the peptide may be useful as a vaccine.
Immunogenic amounts of the antigen can be determined using standard
procedures.
Briefly, various concentrations of a putative specific immunoreactive peptides
or polypeptides
may be prepared, administered to an animal, such as a human, and the
immunological response
(e.g., the production of antibodies or cell-mediated response) of an animal to
each concentration
determined. The pharmaceutically acceptable carrier in the vaccine can
comprise saline or other
suitable carriers (Amon, R. (Ed.), 1987, Synthetic Vaccines 1:83-92, CRC
Press, Inc., Boca
Raton, Florida). An adjuvant can also be a part of the carrier of the vaccine,
in which case it can
be selected by standard criteria based on the antigen used, the mode of
adininistration and the
subject (Amon, 1987). Methods of administration can be by oral or sublingual
means, or by
injection, depending on the particular vaccine used and the subject to whom it
is administered.
In an embodiment, the protein comprising at least one glycoprotein on the
exosporium of
the Bacillus anthracis spore where the glycoprotein coinprises at least one
lectin-binding sugar
may comprise a variant. Spore-specific sugars (rhamnose, 3-0-inethyl rhamnose
and
galactosamine) not found in vegetative cells ofB. antlaracis that are distinct
from the spore sugars
found in related organisms have been found (Fox et al.., 1993; Wunschel et
al., 1994). It has been
directly demonstrated that the anthrax spore is surrounded by carbohydrate.
In an embodiment, the peptide may comprise a Bcl-like peptide. For example,
the
glycoprotein BcIA has a region of tandem repeats as are found in collagen
(Bacillus, collagen-lilce
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protein azzthracis) which consists of approximately 90% carbohydrate
(Sylvester et al., 2002).
Bc1A is localized to the exosporium nap as demonstrated by monoclonal antibody
labeling
(Sylvester et al, 2002). The spore-specific sugars were subsequently
demonstrated to be
components of a glycoprotein Bc1A (Daubenspeck et al., 2004). The operon
coding for Bc1A
synthesis was found, and a second glycoprotein ExsH having tandem repeats was
demonstrated to
be present in B. cereus and B. thuz=izzgiensis (Garcia Patronne, and
Tandecarz, 1995; Todd et al.,
2003).
The peptide backbone of Bc1A has a predicted molecular weight (MW) of
approximately
39-kDa, but the intact protein migrates with an apparent mass of>250-1cDa, for
the Sterne strain,
which is consistent with the protein being heavily glycosylated. There is
considerable size
heterogeneity among the BclA proteins due to different nuinbers of GPT repeats
and
[GPT]5GDTGTT repeats in the protein. The latter 21 amino acid repeat has been
named "the
Bc1A repeat". These repeats are the primary anchor point for rhamnose-
oligosaccharides within
'Bc1A (Sylvestre et al., 2003).
In addition to the known glycoproteins on the exosporium of the Bacillus
anthracis spore,
where the glycoprotein comprises at least one lectin-binding sugar, there are
protein variants
which may also function in the disclosed methods and compositions. In certain
embodiments, the
variants are substitutional, insertional, truncational or deletional variants.
Protein variants and derivatives are well understood to those of skill in the
art and in can
involve amino acid sequence modifications. For example, amino acid sequence
modifications
typically fall into one or more of four classes: substitutional, insertional,
truncational or
deletional variants. Insertions include amino and/or carboxyl terminal fusions
as well as
intrasequence insertions of single or multiple amino acid residues. Insertions
ordinarily will be
smaller insertions than those of anlino or carboxyl terminal fusions, for
example, on the order of
one to four residues. Immunogenic fusion protein derivatives, are made by
fiising a polypeptide
sufficiently large to confer immunogenicity to the target sequence by cross-
linking in vitro or by
recoinbinant cell culture transformed with DNA encoding the fusion.
Truncations are
characterized by the removal of amino acids from the C-terminus or N-temzinus
of the full length
protein. Deletions are characterized by the removal of one or more amino acid
residues from the
protein sequence. Typically, no more than about from 2 to 6 residues are
deleted at any one site
within the protein molecule. These variants ordinarily are prepared by site
specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing DNA encoding
the variant,
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and thereafter expressing the DNA in recoinbinant cell culture. Techniques for
making
substitution mutations at predetermined sites in DNA having a lcnown sequence
are well known,
for example M13 primer mutagenesis and PCR mutagenesis. Amino acid
substitutions are
typically of single residues, but can occur at a number of different locations
at once; insertions
usually will be on the order of about from 1 to 10 amino acid residues; and
deletions will range
about from 1 to 30 residues. Deletions or insertions preferably are made in
adjacent pairs, i.e. a
deletion of 2 residues or insertion of 2 residues. Substitutions, truncations,
deletions, insertions
or any combination thereof may be combined to arrive at a final construct. The
mutations must
not place the sequence out of reading frame and preferably will not create
complementary regions
that could produce secondary mRNA structure. Substitutional variants are those
in which at least
one residue has been removed and a different residue inserted in its place.
Such substitutions
generally are made in accordance with the types of substitutions shown in
Table 2 and are
referred to as conservative substitutions.
TABLE 2:Amino Acid Substitutions
Original Exemplary Conservative
Residue Substitutions, others are known in
the art.
Ala Ser
Arg Lys, Gln
Asn Gln; His
Asp Glu
Cys Aer
Gln Asn, Lys
Glu Asp
Gly Pro
His Asn;Gln
Ile Leu; Val
Leu Ile; Val
Lys; Arg; Gln
Met Leu; Ile
Phe Met; Leu; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp; Phe
Val Ile; Leu
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Substantial changes in function or immunological identity are made by
selecting
substitutions that are less conservative than those in Table 2, i.e.,
selecting residues that differ
more significantly in their effect on maintaining (a) the structure of the
polypeptide baclcbone in
the area of the substitution, for example as a sheet or helical conformation,
(b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain. The
substitutions which in general are expected to -produce the greatest changes
in the protein
properties will be those in which (a) a hydrophilic residue, e.g. seryl or
threonyl, is substituted for
(or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a cysteine
or proline is substituted for (or by) any other residue; (c) a residue having
an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted
for (or by) one not having a side chain, e.g., glycine, in this case, (e) by
increasing the number of
sites for sulfation and/or glycosylation.
For example, the replacement of one amino acid residue with anotller that is
biologically
and/or chemically similar is known to those skilled in the art as a
conservative substitution. For
example, a conservative substitution would be replacing one hydrophobic
residue for another, or
one polar residue for another. The substitutions include combinations such as,
for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; aud Phe, Tyr. Such
conservatively
substituted variations of each explicitly disclosed sequence are included
'within the mosaic
polypeptides provided herein. Substitutional or deletional mutagenesis may be
employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or 0-glycosylation (Ser or
Thr). Deletions of
cysteine or other labile residues also may be desirable. Deletions or
substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by deleting one of
the basic residues or
substituting one by glutaminyl or histidyl residues.
The polypeptides of the present invention may include post-translational
modifications.
In an embodiment, certain post-translational derivatizations are the result of
the action of
recombinant host cells on the expressed polypeptide. Glutaminyl and
asparaginyl residues are
frequently post-translationally deamidated to the corresponding glutamyl aiid
asparyl residues.
Alternatively, these residues are deamidated under mildly acidic conditions.
Other post-
translational modifications include hydroxylation of proline and lysine,
phosphorylation of
hydroxyl groups of seryl or threonyl residues, methylation of the o-amino
groups of lysine,
arginine, and histidine side chains (T.E. Creighton, Proteins: Stiucture and
Molecular Properties,
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W. H. Freeman & Co., San Francisco pp 79-86 (1983)), acetylation of the N-
terminal amine and,
in some instances, amidation of the C-terminal carboxyl.
In an embodiment, the variants and derivatives of the disclosed proteins is
through
defining the variants and derivatives in teims of homology/identity to
specific lcnown sequences.
Those of skill in the art readily understand how to determine the homology
and/or percent
identity of two proteins. For example, the homology can be calculated after
aligning the two
sequences so that the homology is at its highest level. Another way of
calculating homology can
be performed by published algorithms. Optimal alignment of sequences for
comparison may be
conducted by the local homology algorithm of Smith and Waterman, 1981, Adv.
Appl. Math. 2:
482, by the homology aligmnent algorithm of Needleman and Wunsch (1970, J. MoL
Biol. 48:
443 (1970)), by the search for similarity method of Pearson and Lipman, (Proc.
Natl. Acad. Sci.
U.S.A. 85: 2444 (1988), by computerized implementations ofthese algorithms
(GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group,
575 Science Dr., Madison, WI), or by inspection. The same types of homology
can be obtained
for nucleic acids (Zuker, M., 1989, Science 244:48-52; Jaeger et al., 1989,
Proc. Natl. Acad. Sci.
USA, 86:7706-7710; Jaeger et al., 1989, Methods Enzymol., 183:281-306) which
are herein
incorporated by reference for at least material related to nucleic acid
alignment. In an
embodiment, the description of conservative mutations and homology can be
combined together
in any combination, such as embodiments that have at least 80% homology to a
particular
sequence wherein the variants are conservative mutations.
As this specification discusses various proteins and protein sequences it is
understood that
the nucleic acids that can encode those protein sequences are also disclosed.
This would include
all degenerate sequences related to a specific protein sequence, i.e. all
nucleic acids having a
sequence that encodes one particular protein sequence as well as all nucleic
acids, including
degenerate nucleic acids, encoding the disclosed variants and derivatives of
the protein
sequences. Thus, while each particular nucleic acid sequence may not be
written out herein, it is
understood that each and every sequence is in fact disclosed and described
herein through the
disclosed protein sequence. For example, certain of the nucleic acid sequences
sequences of SEQ
ID NO: 1-26 can encode for specific protein sequences as set forth in the
sequences of SEQ ID
NO: 1-26.
In an embodiment, amino acid and peptide analogs can be incorporated into the
disclosed
compositions. For example, there are numerous D ainino acids or amino acids
which have a
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different functional substituent than the amino acids shown in Table 1. In an
embodiment, the
peptides may comprise the opposite stereo isomers of naturally occurring
peptides, as well as the
stereo isomers of peptide analogs. These amino acids can readily be
incorporated into
polypeptide chains by charging tRNA molecules with the amino acid of choice
and engineering
genetic constructs that utilize amber codons to insert the analog amino acid
into a peptide chain
in a site specific way (Thorson et al., 1991, Methods in Molec. Biol. 77:43-
73; Zoller, 1992,
Current Opinion in Biotechnology, 3:348-354; lbba,1995, Biotechnology &
Genetic Engineering
Reviews 13:197-216; Cahill et al., 1989, TIBS, 14(10):400-403; Benner, 1994,
TIBS Tech,
12:158-163; lbba and Hennecke, 1994, Bio/technology, 12:678-682; all of which
are herein
incorporated by reference at least for material related to amino acid
analogs).
In an embodiment, the compounds of the present invention may include molecules
that
resemble peptides, but which are not connected via a natural peptide linkage.
For example,
linkages for amino acids or amino acid analogs can include [(CH2NH)--], [--
(CH2S)--], [--(CH2--
CH2) --], [--(CH=CH)--] [(cis and trans)], [--(COCH2) --], [--(CH(OH)CH2)--],
and [-
(CHH2SO)-] (Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,
Peptides, and
Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola,
A. F., Vega Data
(March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general
review); Morley, Trends
Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-
185 (1979) [--
(CH2NH)--, (CH2CH2)--]; Spatola et al. Life Sci 38:1243-1249 (1986) [--(CH HZ)-
-(S)]; Hann J.
Chem. Soc Perlcin Trans. I 307-314 (1982) [--(CH-CH)--, cis and trans];
Almquist et al. J. Med.
Chem. 23:1392-1398 (1980) [--(COCH2)--]; Jennings-White et al. Tetrahedron
Lett 23:2533
(1982) [--(COCH2)--]; Szelke et al. European Appln, EP 45665 CA (1982):
97:39405 (1982) [--
(CH(OH)CH2)--]; Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) [--(C(OI-
1)CH2)--]; and
Hruby Life Sci 31:189-199 (1982) [--(CH2)--(S)--]; each of which is
incorporated herein by
reference. A particularly preferred non-peptide linlcage is -[--(CH2NH)--]. It
is understood that
peptide analogs can have more tlian one atom between the bond atoms, such as b-
alanine, g-
aminobutyric acid, and the lilce. Amino acid analogs and analogs and peptide
analogs often have
enhanced or desirable properties, such as, more economical production, greater
chemical
stability, enhanced pharmacological properties (half-life, absorption,
potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological activities), reduced
antigenicity, and
others. D-amino acids can be used to generate more stable peptides, because D
ainino acids are
not recognized by peptidases and such. Systeniatic substitution of one or more
amino acids of a
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consensus sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can
be used to generate more stable peptides. Cysteine residues can be used to
cyclize or attach two
or more peptides together. This can be beneficial to constrain peptides into
particular
conformations. (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61:387).
8. Nucleic acids
As vaccines can consist of nucleic acids, there are a variety of molecules
disclosed herein
that are nucleic acid based, including the nucleic acids that encode for at
least one glycoprotein
from an extract of the exosporium of the Bacillus arathracis spore by
absorption of the extract to
lectin as well as any other proteins disclosed herein and variants and
fragments of such
polypeptides and/or proteins. In an embodiment, the nucleic acids used in the
vaccines of the
present invention may comprose nucleotides, nucleotide aiialogs, or nucleotide
substitutes. Non-
limiting examples of these and other molecules are discussed herein.
A nucleotide is a molecule that contains a base moiety, a sugar moiety and a
phosphate
moiety. Nucleotides can be linked together through their phosphate moieties
and sugar moieties
creating an intemucleoside linkage. The base moiety of a nucleotide can be
adenin-9-yl (A),
cytosin-l-yl (C), guanin-9-yl (G), uracil-l-yl (U), and thymin-l-yl (T). The
sugar moiety of a
nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide
is pentavalent
phosphate. An non-limiting example of a nucleotide would be 3'-AMP (3'-
adenosine
monophosphate) or 5'-GMP (5'-guanosine monophosphate). It is understood for
example that
when a vector is expressed in a cell the expressed mRNA will typically be made
up of A, C, G,
and U. Lilcewise, it is understood that if, for example, an antisense molecule
is introduced into a
cell or cell environment through for example exogenous delivery, it is
advantageous that the
antisense molecule be made up of nucleotide analogs that reduce the
degradation of the antisense
molecule in the cellular environment.
In certain embodiments, the nucleotide vaccines of the present invention may
comprise at
least one of a nucleotide analog, a nucleotide substitute, or a conjugated
nucleotide. A nucleotide
analog is a nucleotide which contains some type of modification to either the
base, sugar, or
phosphate moieties. Modifications to nucleotides are well known in the art and
would include
for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxantliine, and
2-aminoadenine as well as modifications at the sugar or phosphate moieties.
Nucleotide
substitutes are molecules having similar functional properties to nucleotides,
but whicli do not
contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide
substitutes are
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molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen
manner, but which
are linked together through a moiety other than a phosphate moiety. Nucleotide
substitutes are
able to conform to a double helix type structure when interacting with the
appropriate target
nucleic acid. Other types of molecules may be linked to nucleic acid molecules
to form
conjugates. Conjugates can be chemically linlced to the nucleotide or
nucleotide analogs. Such
conjugates include but are not limited to lipid moieties such as a cholesterol
moiety. (Letsinger et
al., 1989, Proc. Natl. Acad. Sci. USA,86, 6553-6556). A Watson-Crick
interaction is at least
one interaction with the Watson-Crick face of a nucleotide, nucleotide analog,
or nucleotide
substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or
nucleotide substitute
includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide
analog, or
nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based
nucleotide, nucleotide
analog, or nucleotide substitute. A Hoogsteen interaction is the interaction
that talces place on the
Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the
major groove of
duplex DNA. The Hoogsteen face includes the N7 position and reactive groups
(NH2 or 0) at the
C6 position of purine nucleotides.
Embodiments of the present invention also comprise oligonucleotides that are
capable of
interacting as either primers or probes with genes that encode for the
glycoproteins and
polypeptides associated with the glycoproteins of the complexes found in the
B. anthracis spore
as described herein. In certain embodiments the primers are used to support
DNA amplification
reactions. Typically the primers will be capable of being extended in a
sequence specific manner.
Extension of a primer in a sequence specific manner includes any metliods
wlierein the sequence
and/or composition of the nucleic acid molecule to which the primer is
llybridized or otherwise
associated directs or influences the composition or sequence of the product
produced by the
extension of the primer. Extension of the primer in a sequence specific
maiiner therefore
includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA
polymerization,
RNA transcription, or reverse transcription. Techniques and conditions that
amplify the primer
in a sequence specific maimer are preferred. In certain embodiments the
primers are used for the
DNA amplification reactions, such as PCR or direct sequencing. It is
understood that in certain
embodiments the primers can also be extended using non-enzymatic techniques,
where for
example, the nucleotides or oligonucleotides used to extend the primer are
modified such that
they will cheniically react to extend the priiner in a sequence specific
manner. Typically the
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disclosed primers hybridize with the nucleic acid or region of the nucleic
acid or they hybridize
with the complement of the nucleic acid or complement of a region of the
nucleic acid.
In an embodiment, the compositions are formiilated for delivery to a cell,
either in vivo of
in vitro. There are a number of compositions and methods which can be used to
deliver nucleic
acids to cells, either in vitro or in vivo. These methods and compositions can
largely be broken
down into two classes: viral based delivery systems and non-viral based
delivery systems. For
example, the nucleic acids can be delivered by a number of direct delivery
systems such as,
electroporation, lipofection, calcium phosphate precipitation, plasmids, viral
vectors, viral
nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of
genetic material in cells or
carriers such as cationic liposomes. Appropriate means for transfection,
including viral vectors,
chemical transfectants, or physico-mechanical methods such as electroporation
and direct
diffusion of DNA (Wolff, J. A., et al., 1990, Science, 247, 1465-1468; Wolff,
J. A., 1991,
Nature, 352, 815-818). Sucli methods are well known in the art and readily
adaptable for use with
the compositions and methods described herein. In certain cases, the methods
will be modified to
specifically function with large DNA molecules. Further, these methods can be
used to target
certain diseases and cell populations by using the targeting characteristics
of the carrier.
In an embodiment, the present invention may comprise the use of transfer
vectors to
deliver genes into cells (e.g., a plasmid), or as part of a general strategy
to deliver genes, e.g., as
part of recombinant retrovirus or adenovirus (Ram et al., 1993, Cancer Res.
53:83-88). As used
herein, plasmid or viral vectors are agents that transport the nucleic acid of
interest into a cell
witliout degradation. The transfer vectors may comprise a promoter yielding
expression of the
gene of interest in the cells into which it is delivered. In some embodiments
the vectors are
derived from either a virus or a retrovirus. Viral vectors that may be used to
deliver the DNA
constructs of the present invention to cells may comprise Adenovirus, Adeno-
associated virus,
Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus,
Sindbis and other
RNA viruses, including these vintses with the HIV baclcbone. Also included are
any viral
families which share the properties of these viruses which malce them suitable
for use as vectors.
For example, retroviruses, including Murine Maloney Leukemia virus, MMLV, and
retroviruses
that express the desirable properties of MMLV as a vector may be used to
deliver the DNA
constructs of the present invention to cells. Retroviral vectors are able to
carry a larger genetic
payload, i.e., a transgene or marlcer gene, tllan other viral vectors, and for
this reason are a
conlnionly used vector. However, they are not as useful in non-proliferating
cells. Adenovirus
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vectors are relatively stable and easy to work with, have high titers, and can
be delivered in
aerosol formulation, and can transfect non-dividing cells. Pox viral vectors
are large and have
several sites for inserting genes, they are thermostable and can be stored at
room temperature. In
an embodiment, a viral vector which has been engineered so as to suppress the
immune response
of the host organism, elicited by the viral antigens may be used such as
vectors that carry coding
regions for Interleukin 8 or 10.
Viral vectors can have higher transaction (ability to introduce genes)
abilities than
chemical or physical methods to introduce genes into cells. Typically, viral
vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase III
transcript, inverted
terminal repeats necessary for replication and encapsidation, and promoters to
control the
transcription and replication of the viral genome. When engineered as vectors,
viruses typically
have one or more of the early genes removed and a gene or. gene/promoter
cassette is inserted into
the viral genome in place of the removed viral DNA. Constructs of this type
can carry up to
about 8 kb of foreign genetic material. The necessary functions of the removed
early genes are
typically supplied by cell lines which have been engineered to express the
gene products of the
early genes in trans.
i. Retroviral Vectors
In an embodiment, a retrovirus is used to deliver the nucleic acid molecules
of the present
invention to a cell. A retrovirus is an animal virus belonging to the virus
fainily of Retroviridae,
including any types, subfamilies, genus, or tropisms. ' Examples of methods
for using retroviral
vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and
4,980,286; PCT
applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932
(1993)); the
teachings of which are incorporated herein by reference.
A retrovirus is essentially a package which has paclced into it nucleic acid
cargo. The
nucleic acid cargo carries with it a packaging signal, which ensures that the
replicated daughter
molecules will be efficiently packaged within the package coat. In addition to
the package signal,
there are a number of molecules wliich are needed in cis, for the replication,
and paclcaging of the
replicated virus. Typically a retroviral genome, contains the gag, pol, and
env genes which are
involved in the malcing of the protein coat. It is the gag, pol, and env genes
which are typically
replaced by the foreign DNA that it is to be transferred to the target cell.
Retrovinis vectors
typically contain a packaging signal for incorporation into the paclcage coat,
a sequence which
signals the start of the gag transcription unit, elements necessary for
reverse transcription,
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including a primer binding site to bind the tRNA primer of reverse
transcription, terminal repeat
sequences that guide the switch of RNA strands during DNA synthesis, a purine
rich sequence 5'
to the 3' LTR that serve as the priming site for the synthesis of the second
strand of DNA
synthesis, and specific sequences near the ends of the LTRs that enable the
insertion of the DNA
state of the retrovirus to insert into the host genome. The removal of the
gag, pol, and env genes
allows for about 8 kb of foreign sequence to be inserted into the viral
genome, become reverse
transcribed, and upon replication be packaged into a new retroviral particle.
This amount of
nucleic acid is sufficient for the delivery of a one to many genes depending
on the size of each
transcript. It is preferable to include either positive or negative selectable
marlcers along with
other genes in the insert.
Since the replication machinery and packaging proteins in most retroviral
vectors have
been removed (gag, pol, and env), the vectors are typically generated by
placing them into a
packaging cell line. A packaging cell line is a cell line which has been
transfected or transformed
with a retrovirus that contains the replication and packaging machinery, but
lacks any packaging
signal. When the vector carrying the DNA of choice is transfected into these
cell lines, the vector
containing the gene of interest is replicated and packaged into new retroviral
particles, by the
machinery provided in cis by the helper cell. The genomes for the machinery
are not packaged
because they lack the necessary signals.
ii. Adenoviral Vectors
In an embodiment, an adenovirus vector is used to deliver the nucleic acid
molecules of
the present invention to cells. Replication-incompetent adenoviruses are
currently available
efficient gene transfer vehicles for both in vitro and in vivo deliveries
(Lukashok, S. A., and M.
S. Horwitz. 1998. Current Clinical Topics in Infectious Diseases 18:286-305).
Adenovirus-,
vectored recoinbinant vaccines expressing a wide array of antigens have been
constructed and
protective immunities against different pathogens have been demonstrated in
animal models
(Lubeck, M. D., et al. 1997. Nat Med 3:651-8) (Shi, Z., et al., 2001, J
Viro175:11474-82; Shiver,
J. W., et al., 2002, Nature 415:331-5; Tan, Y., et al., 2003, Hum Gene Ther
14:1673-82).
The construction of replication-defective adenoviruses has been described
(Berkner et al.,
J. Virology, 1987, 61:1213-1220; Massie et al., 1986, Mol. Cell. Biol. 6:2872-
2883; Haj-
Ahmad et al., 1986, J. Virology 57:267-274; Davidson et al., 1987, J. Virology
61:1226-1239;
Zhang, 1993, BioTechniques 15:868-872). The benefit of the use of these
viruses as vectors is
that they are limited in the extent to which they can spread to other cell
types, since they can
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replicate within an initial infected cell, but are unable to form new
infectious viral particles.
Recombinant adenoviruses have been shown to achieve high efficiency gene
transfer after direct,
in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS
parenchyma and a
number of other tissue sites (Morsy, 1993, J. Clin. Invest. 92:1580-1586;
Kirshenbaum, 1993, J.
Clin. Invest. 92:381-387; Roessler, 1993, J. Clin. Invest. 92:1085-1092;
Moullier, 1993, Nature
Genetics 4:154-159; La Salle, Science, 1993, 259:988-990; Gomez-Foix, 1992, J.
Biol. Chem.
267:25129-25134; Rich, 1993, Human Gene Therapy 4:461-476; Zabner, 1994,
Nature Genetics
6:75-83; Guzman, 1993, Circulation Research 73:1201-1207; Bout, 1994, Huinan
Gene Therapy
5:3-10; Zabner, 1993, Cel175:207-216; Caillaud, 1993, Eur. J. Neuroscience
5:1287-1291; and
Ragot, 1993, J. Gen. Virology 74:501-507). Recoinbinant adenoviruses achieve
gene
transduction by binding to specific cell surface receptors, after which the
virus is internalized by
receptor-mediated endocytosis, in the same manner as wild type or replication-
defective
adenovirus (Chardonnet and Dales, 1970, Virology 40:462-477); Brown and
Burlingham, 1973,
J. Virology 12:386-396); Svensson and Persson, 1985, J. Virology 55:442-449);
Seth, et al.,
1984, J. Virol. 51:650-655); Seth, et al., 1984, Mol. Cell. Biol. 4:1528-
1533); Varga et al.,
1991, J. Virology 65:6061-6070); Wiclcham et al., 1993, Cel173:309-319).
The viral vector can be one based on an adenovirus which has had the El gene
removed.
The El gene is necessary for viral replication and expression. However, E1-
deleted viruses can
be propagated in cell lines that provide El in tram, such as 293 cells (Graham
arid Prevec, 1995,
Mol. Biotechnol. 3:207-220). In another embodiment, both the El and E3 genes
are removed
from the adenovirus genome. The E3 region is involved in blocking the immune
response to the
infected cell.
In yet another embodiment, alternative serotype adenoviral vectors, such as
human Ad3 5
or Ad7 to which the majority of human populations have very low pre-existing
immunity could
be used (31, 46). Also, adenoviral vectors derived from animals such as ovine
and chimpanzee
adenoviruses could also be used as alternative vaccine delivery vectors
(Farina, S. F. et al. J Virol
75:11603-13; Hofinann, C. et al. 1999. J Virol 73:6930-6).
iii. Adeno-associated viral vectors
In an embodiment, an Adeno-associated viral vector is used to deliver the
nucleic acid
molecules of the present invention to cells. Another type of viral vector is
based on an adeno-
associated virus (AAV). This defective parvovirus is a,preferred vector
because it can infect
many cell types and is nonpathogenic to humans. AAV type vectors can transport
about 4 to 5 lcb
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and wild type AAV is known to stably insert into cliromosoine 19. Vectors
which contain this
site specific integration property are preferred. An especially preferred
embodiment of this type
of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which
can contain the
herpes simplex virus thynlidine kinase geiie, HSV-tlc, and/or a marker gene,
such as the gene
encoding the green fluorescent protein, GFP. In another type of AAV virus, the
AAV contains a
pair of inverted terminal repeats (ITRs) which flank at least one cassette
containing a promoter
which directs cell-specific expression operably linked to a heterologous gene.
Heterologous in
this context refers to any nucleotide sequence or gene which is not native to
the AAV or B 19
parvovirus. Typically the AAV and B 19 coding regions have been deleted,
resulting in a safe,
noncytotoxic vector. The AAV ITRs, or modifications thereof, confer
infectivity and site-
specific integration, but not cytotoxicity, and the promoter directs cell-
specific expression. U.S.
Patent No. 6,261, 834 is lierein incorporated by reference for material
related to the AAV vector.
In certain embodiments, the inserted genes in viral and retroviral vectors
will contain p'romoters,
and/or enliancers to help control the expression of the desired gene product.
iv. Large payload viral vectors
In yet another embodiment, a large payload viral vector, such as a herpes
virus vector, is
used to deliver the nucleic acid molecules of the present invention to cells.
Molecular genetic
experiments with large human herpesviruses have provided a means whereby large
heterologous
DNA fragments can be cloned, propagated and established in cells permissive
for infection with
herpesviruses (Sun et al., 1994, Nature genetics 8: 33-41; Cotter and
Robertson, 1999, Curr.
Opin. Mol. Ther., 5: 633-644). These large DNA viruses (herpes simplex virus
(HSV) and
Epstein-Barr virus (EBV), have the potential to deliver fragments of human
heterologous DNA >
150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in
the infected B-
cells as episomal DNA. Individual clones carried human genomic inserts up to
330 lcb appeared
genetically stable. The maintenance of these episomes requires a specific EBV
nuclear protein,
EBNA1, constitutively expressed during infection with EBV. Additionally, these
vectors can be
used for transfection, where large amounts of protein can be generated
transiently in vitro.
Herpesvirus amplicon systems are also being used to package pieces of DNA >
220 lcb and to
infect cells that can stably maintain DNA as episomes. In other embodiments,
replicating and
host-restricted non-replicating vaccinia virus vectors may also be used.
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v. Non-nucleic acid based systems
The nucleic acid molecules of the present invention can be delivered to the
target cells in
a variety of ways. For example, in certain embodiments, the compositions may
be delivered
through electroporation, or through lipofection, or through calcium phosphate
precipitation. The
delivery mechanism chosen will depend in part on the type of cell targeted and
whether the
delivery is occurring in vivo or in vitro.
Thus, the compositions can comprise, in addition to the disclosed viruses or
vectors for
example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA,
DOPE,
DC-cholesterol) or aiiionic liposomes. Liposomes can further comprise proteins
to facilitate
targeting a particular cell, if desired. Administration of a composition
comprising a compound
and a cationic liposome can be administered to the blood afferent to a target
organ or inhaled into
the respiratory tract to target cells of the respiratory tract (see, e.g.,
Brigham et al., 1989, Am. J.
Resp. Cell. Mol. Biol. 1:95-100); Felgner et a1.,1987, Proc. Natl. Acad. Sci
USA 84:7413-7417
); U.S. Pat. No. 4,897,355). Furthermore, the compound can be administered as
a component of
a microcapsule that can be targeted to specific cell types, such as
macrophages, or where the
diffusion of the compound or delivery of the compound from the microcapsule is
designed for a
specific rate or dosage.
In the methods described above which include the adininistration and uptalce
of
exogenous DNA into the cells of a subject (i.e., gene transduction or
transfection), delivery of the
compositions to cells can be via a variety of mechanisms. As one example,
delivery can be via a
liposome, using commercially available liposome preparations such as
LIPOFECTIN,
LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc.
Hilden,
Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other
liposomes developed according to procedures standard in the art. In addition,
the disclosed
nucleic acid or vector can be delivered in vivo by electroporation, the
technology for which is
available from Genetronics, Inc. (San Diego, CA) as well as by means of a
SONOPORATION
machine (ImaRx Pharmaceutical Corp., Tucson, AZ).
The materials may be in solution, suspension (for example, incorporated into
microparticles, liposomes, or cells). These may be targeted to a particular
cell type via
antibodies, receptors, or receptor ligands. The following references are
exainples of the use of
this technology to target specific proteins to tuinor tissue (Senter, et al.,
1991, Bioconjugate
Chem., 2:447-451; Bagshawe, K.D., 1989, Br. J. Cancer, 60:275-281; Bagshawe,
et a1.,1988, Br.
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J. Cancer, 58:700-703; Senter, et al., 1993, Bioconjugate Chem., 4:3-9;
Battelli, et al.,1992,
Cancer Immunol. Immunother., 35:421-425; Pietersz and McKenzie, 1992,
Immunolog.
Reviews, 129:57-80); and Roffler, et al., 1991, Biochem. Phamlacol, 42:2062-
2065). These
techniques can be used for a variety of other specific cell types. Vehicles
such as "stealth" and
other antibody conjugated liposomes (including lipid mediated drug targeting
to colonic
carcinoma), receptor mediated targeting of DNA through cell specific ligands,
lymphocyte
directed tumor targeting, and higlily specific therapeutic retroviral
targeting of murine glioma
cells in vivo (Hughes et al., 1989, Cancer Research, 49:6214-6220; and
Litzinger and Huang,
1992, Biochimica et Biophysica Acta, 1104:179-187). In general, receptors are
involved in
pathways of endocytosis, either constitutive or ligand induced. These
receptors cluster in
clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass
througli an acidified
endosome in which the receptors are sorted, and then either recycle to the
cell surface, become
stored intracellularly, or are degraded in lysosomes. The internalization
pathways serve a variety
of functions, such as nutrient uptake, removal of activated proteins,
clearance of macromolecules,
opportunistic entry of viruses and toxins, dissociation and degradation of
ligand, and
receptor-level regulation. Many receptors follow more than one intracellular
pathway, depending
on the cell type, receptor concentration, type of ligand, ligand valency, and
ligand concentration.
Molecular and cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown
and Greene, 1991, DNA and Cell Biology 10:6, 399-409).
Nucleic acids that are delivered to cells which are to be integrated into the
host cell
genome, typically contain integration sequences. These sequences are often
viral related
sequences, particularly when viral based systems are used. These viral
integration systems can
also be incorporated into nucleic acids which are to be delivered using a non-
nucleic acid based
system of deliver, such as a liposome, so that the nucleic acid contained in
the delivery system
can be come integrated into the llost genome.
Other general techniques for integration into the host genome include, for
example,
systems designed to promote homologous recombination with the host genome.
These systems
typically rely on sequence flanlcing the nucleic acid to be expressed that has
enough homology
with a target sequence within the host cell genome that recoinbination between
the vector nucleic
acid and the target nucleic acid talces place, causing the delivered nucleic
acid to be integrated
into the host genome. These systems and the methods necessary to promote
homologous
recombination are lcnown to those of skill in the art.
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In an embodiment, the nucleic acid molecules can be administered in a
pharmaceutically
acceptable carrier and can be delivered to the subjects' cells in vivo and/or
ex vivo by a variety of
mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion,
intramuscular
injection of DNA via a gene gun, endocytosis and the like). If ex vivo methods
are employed,
cells or tissues can be removed and maintained outside the body according to
standard protocols
well known in the art. The compositions can be introduced into the cells via
any gene transfer
mechanism, such as, for example, calcium phosphate mediated gene delivery,
electroporation,
microinjection or proteoliposomes. The transduced cells can then be infused
(e.g., in a
pharmaceutically acceptable carrier) or homotopically transplanted back into
the subject per
standard methods for the cell or tissue type. Standard methods are lcnown for
transplantation or
infusion of various cells into a subject.
(e) Expression systems
In an embodiment, the nucleic acids that are delivered to cells may contain
expression
controlling systems. For example, the inserted genes in viral and retroviral
systems usually
contain promoters, and/or enhancers to help control the expression of the
desired gene product.
A promoter is generally a sequence or sequences of DNA that function when in a
relatively fixed
location in regard to the transcription start site. A promoter contains core
elements required for
basic interaction of RNA polymerase and transcription factors, and may contain
upstream
elements and response elements.
In certain embodiinents, promoters controlling transcription from vectors in
mammalian
host cells may be obtained from various sources, for example, the genomes of
viruses such as:
polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus
and most
preferably cytomegalovirus, or from heterologous inainmalian promoters, e.g.
beta actin
promoter. The early and late promoters of the SV40 virus are conveniently
obtained as an SV40
restriction fragment which also contains the SV40 viral origin of replication
(Fiers et al., Nature,
273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is
conveniently
obtained as a HindIII E restriction fragment (Greenway, P.J. et al., Gene 18:
355-360 (1982)).
Of course, promoters from the host cell or related species also are useful
herein.
As used herein, an enhancer generally refers to a sequence of DNA that
fiulctions at no
fixed distance from the transcription start site and can be either 5'
(Laimins, L. et al., Proc. Natl.
Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108
(1983)) to the
transcription unit. Furthermore, enhancers can be within an intron (Banerji,
J.L. et al., Cell 33:
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729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et
al., Mol. Cell Bio. 4:
1293 (1984)). They are usually between 10 and 300 bp in length, and they
function in cis.
Enhancers f unction to increase transcription from nearby promoters. Enhancers
also often
contain response elements that mediate the regulation of transcription.
Promoters can also
contain response elements that mediate the regulation of transcription.
Enhancers often
determine the regulation of expression of a gene. While many enhancer
sequences are now
known from mammalian genes (globin, elastase, albumin, -fetoprotein and
insulin), typically one
will use an enhancer from a eukaryotic cell virus for general expression.
Preferred examples are
the SV40 enhancer on the late side of the replication origin (bp 100-270), the
cytomegalovirus
early promoter enhancer, the polyoma enhancer on the late side of the
replication origin, and
adenovirus enhancers.
In certain embodiments, the promoter and/or enhancer may be specifically
activated either
by light or specific chemical events which trigger their function. Systems can
be regulated by
reagents such as tetracycline and dexamethasone. There are also ways to
enhance viral vector
gene expression by exposure to irradiation, such as gamma irradiation, or
allcylating
chemotherapy drugs.
Also, in certain embodiments, the promoter and/or enhancer region can act as a
constitutive promoter and/or enhancer to maximize expression of the region of
the transcription
unit to be transcribed. In certain constructs the promoter and/or enhaticer
region be active in all
eukaryotic cell types, even if it is only expressed in a particular type of
cell at a particular time. A
preferred promoter of this type is the CMV promoter (650 bases). Other
preferred promoters are
SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector
LTF.
It has been shown that all specific regulatory elements can be cloned and used
to construct
expression vectors that are selectively expressed in specific cell types such
as melanoma cells.
The glial fibrillary acetic protein (GFAP) promoter has been used to
selectively express genes in
cells of glial origin.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human
or nucleated cells) may also contain sequences necessary for the termination
of transcription
which may affect mRNA expression. These regions are transcribed as
polyadenylated segments
in the untranslated portion of the mRNA encoding tissue factor protein. The 3'
untranslated
regions also include transcription termination sites. It is preferred that the
transcription unit also
contain a polyadenylation region. One benefit of this region is that it
increases the likelihood that
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the transcribed unit will be processed and transported like mRNA. The
identification and use of
polyadenylation signals in expression constructs is well established. It is
preferred that
homologous polyadenylation signals be used in the transgene constracts. In
certain transcription
units, the polyadenylation region is derived from the SV40 early
polyadenylation signal and
consists of about 400 bases. It is also preferred that the transcribed units
contain other standard
sequences alone or in combination with the above sequences improve expression
from, or
stability of, the construct.
(f) Markers
In certain embodinlents, the viral vectors can include nucleic acid sequence
encoding a
marker product. This marker product is used to determine if the gene has been
delivered to the
cell and once delivered is being expressed. Preferred marker genes are the E.
Coli lacZ gene,
which encodes 13-galactosidase, and green fluorescent protein.
In some embodiments the marker may be a selectable marker. Examples of
suitable
selectable markers for manunalian cells are dihydrofolate reductase (DHFR),
thyinidine kinase,
neomycin, neomycin analog G418, hydromycin, and puromycin. When such
selectable marlcers
are successfully transferred into a mammalian host cell, the transformed
mammalian host cell can
survive if placed under selective pressure. There are two widely used distinct
categories of
selective regimes. The first category is based on a cell's metabolism and the
use of a mutant cell
line which lacks the ability to grow independent of a supplemented media. Two
examples are:
CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow
without the
addition of such nutrients as thymidine or hypoxanthine. Because these cells
lack certain genes
necessary for a complete nucleotide synthesis pathway, they camzot survive
unless the missing
nucleotides are provided in a supplemented media. An alternative to
supplementing the media is
to introduce an intact DHFR or TK gene into cells lacking the respective
genes, thus altering their
growth requirements. Individual cells which were not transformed with the DHFR
or TK gene
will not be capable of survival in non-supplemented media.
The second category is dominant selection which refers to a selection scheine
used in any
cell type and does not require the use of a mutant cell line. These schemes
typically use a drug to
arrest growth of a host cell. Those cells wliich have a novel gene would
express a protein
conveying drug resistance and would survive the selection. Examples of such
dominant selection
use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:
327 (1982)),
mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or
hygromycin,
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(Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples
employ bacterial
genes under eukaryotic control to convey resistance to the appropriate drug
G418 or neomycin
(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others
include the neomycin
analog G418 and puramycin.
10. Methods of making the compositions
The compositions disclosed herein and the con-ipositions necessary to perform
the
disclosed methods can be made using any method known to those of skill in the
art for that
particular reagent or compound unless otherwise specifically noted. It is also
understood that
basic recombinant biotechnology methods can be used to produce the nucleic
acids and proteins
disclosed herein.
1. Nucleic acid synthesis
For example, the nucleic acids, such as, the oligonucleotides to be used as
primers can be
made using standard chemical synthesis methods or can be produced using
enzymatic methods or
any other known method. Such methods can range from standard enzymatic
digestion followed
by nucleotide fragment isolation (see for exainple, Sambrook et al.,
Moleculai= Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor,
N.Y., 1989, Chapters 5, 6) to purely synthetic methods, for example, by the
cyanoethyl
phosphoramidite method using a Milligen or Beclcman System 1Plus DNA
synthesizer (for
example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington,
MA or ABI
Mode1380B; Ikuta et al.,1984, Ann. Rev. Biochena. 53:323-356, describing a
phosphotriester and
phosphite-triester methods; and Narang et al.,1980, Methods Enzyrnol., 65:610-
620; describing a
phosphotriester method). Protein nucleic acid molecules can be made using
lcnown methods
(e.g., Nielsen et al., 1994, Bioconjug. Chem. 5:3-7).
2. Peptide synthesis
One method of producing a protein for use as in a B. anthracis vaccine, such
as those
included in the sequences of SEQ ID NO: 1-26 is to link two or more peptides
or polypeptides
together by protein chemistry techniques. For example, peptides or
polypeptides can be
chemically synthesized using currently available laboratory equipment using
either Fmoc
(9-fluorenylmetlzyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry.
(Applied
Biosystems, Inc., Foster City, CA). One slcilled in the art can readily
appreciate that a peptide or
polypeptide corresponding to the disclosed proteins, for example, can be
synthesized by standard
chemical reactions. For example, a peptide or polypeptide can be synthesized
and not cleaved
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from its synthesis resin whereas the other fragnient of.a peptide or protein
can be synthesized and
subsequently cleaved from the resin, thereby exposing a terminal group which
is functionally
blocked on the other fragment. By peptide condensation reactions, these two
fragments can be
covalently j oined via a peptide bond at their carboxyl and amino termini,
respectively, to form an
antibody, or fragment thereof. (Grant GA, 1992, Synthetic Peptides: A User
Guide. W.H.
Freeman and Co., N.Y., 1992; Bodanslcy M and Trost B., Ed., 1993, Principles
of Peptide
Synthesis. Springer-Verlag Inc., NY. Alternatively, the peptide or polypeptide
is independently
synthesized in vivo as described herein. Once isolated, these independent
peptides or
polypeptides may be linked to fonn a peptide or fragment thereof via similar
peptide
condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments allow
relatively
short peptide fragments to be joined to produce larger peptide fragments,
polypeptides or whole
protein domains (Abrahlnsen L et al., 1991, Biochemistry, 30:4151).
Alternatively, native
chemical ligation of synthetic peptides can be utilized to synthetically
construct large peptides or
polypeptides from shorter peptide fragments. This inethod consists of a two
step chemical
reaction (Dawson et al., 1994, Synthesis of Proteins by Native Chemical
Ligation. Science,
266:776-779). The first step is the chemoselective reaction of an unprotected
synthetic
peptide--thioester with another unprotected peptide segment containing an
amino-terminal Cys
residue to give a thioester-linlced intermediate as the initial covalent
product. Without a change
in the reaction conditions, this intermediate undergoes spontaneous, rapid
intramolecular reaction
to form a native peptide bond at the ligation site (Baggiolini M et al., 1992,
FEBS Lett. 307:97-
101; Clark-Lewis I et al., 1994, J.Biol.Chem., 269:16075; Clarlc-Lewis I et
al., 1991,
Biochemistry, 30:3128; Rajarathnam K et al., 1994, Biochemistry 33:6623-30).
Alternatively, unprotected peptide segments are cheinically linlced where the
bond formed
between the peptide segments as a result of the chemical ligation is an
unnatural (non-peptide)
bond (Schnolzer, M et al. , 1992, Science, 256:221). This technique has been
used to synthesize
analogs of protein domains as well as large ainounts of relatively pure
proteins with full
biological activity (deLisle Milton RC et al., 1992, Techniques in Protein
Chemistry IV.
Academic Press, New York, pp. 257-267).
3. Processes for making the compositions
In an einbodiment, the spore surface glycoproteins complexes are produced
after urea
extracted or lysed spores are lectin purified. In an embodiment, the
preparation comprises
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proteins, glycoproteins, oligosaccharides, lipids, or phospholipids that are
produced by lysing the
spore by urea extract or another means of lysis such as sonication but not
limited to the above
listed techniques. In an embodiment, the composition may comprise proteins,
glycoproteins,
polysaccharides, lipids, or phospholipids isolated by electro-elution or size
exclusion
cliromatograplly after the spores have been lysed.
Embodiments of the present invention also comprise processes for malcing the
compositions as well as malcing the intermediates leading to the compositions,
and where
reference to a particular sequence occurs, this is understood as exemplary
only. In an
embodiment, the protein used in the vaccine comprises a sequence that is
encoded by one of the
nucleic acid sequences having the sequence as set forth in any one of the
nucleic acid sequences
of sequences 1-26. There are a variety of methods that can be used for making
these
compositions, such as synthetic chemical methods and standard molecular
biology metliods. It is
understood that the methods of making these and the other disclosed
compositions are
specifically disclosed. For example, in an embodinlent, the protein or
polypeptide of interest is
generated by linking in an operative way a sequence that is encoded by one of
the nucleic acid
sequences having the sequence as set forth in any one of the nucleic acid
sequences of sequences
1-26 to a sequence controlling the expression of the nucleic acid. In an
embodiment, the nucleic
acid sequence may comprise at least 80%, or at least 90%, or at least 95%, or
at least 99%
sequence identity to one of the nucleic acid sequences having the sequence as
set forth in any one
of the nucleic acid sequences of sequences 1-26. Or, a sequence that
hybridizes under stringent
liybridization conditions to one of the nucleic acid sequences having the
sequence as set forth in
any one of the nucleic acid sequences of sequences 1-26 may be used. For
example, in an
embodiment, the present invention comprises an isolated nucleic acid molecule
encoding a lectin-
binding glycoprotein isolated from the exosporium of the Bacillus anthf=acis
spore comprising a
nucleic acid sequence as set forth in SEQ ID NO: 1, SEQ ID. NO: 3, SEQ ID. NO:
5, SEQ ID.
NO: 7, SEQ ID. NO: 9, SEQ ID. NO: 11, SEQ ID. NO: 13, SEQ ID. NO: 15, SEQ ID.
NO: 17,
SEQ ID. NO: 19, SEQ ID. NO: 21, SEQ ID. NO: 23, or SEQ ID. NO: 25.
The polypeptide encoded by the nucleic acid construct may coinprise one of the
polypeptide sequences having the sequence as set forth in any one of the amino
acid sequences of
sequences 1-26, or a fragment of such a protein, or a protein having
conservative amino acid
substitutions. In an embodiment, the amino acid sequence has at least 80%
homology to at least
one of the amino acid sequences as set forth in SEQ ID. NO: 2, SEQ ID. NO: 4,
SEQ ID. NO: 6,
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SEQ ID. NO: 8, SEQ ID. NO: 10, SEQ ID. NO: 12, SEQ TD. NO: 14, SEQ ID. NO: 16,
SEQ ID.
NO: 18, SEQ ID. NO: 20, SEQ ID. NO: 22, SEQ ID. NO: 24, SEQ ID. NO: 26.
In yet another enibodiment, the present invention comprises genetically
modified animals
produced by the process of transfecting a cell within the animal with any of
the nucleic acid
molecules disclosed herein. The animal may be a mammal. In alternate
embodiments, the
mammal may be a mouse, rat, rabbit, cow, sheep, pig, or prinlate.
Alternatively, a genetically
modified animal may be made by adding to the animal any of the cells disclosed
herein.
EXAMPLES
The following examples are put forth so as to provide those of ordinary slcill
in the art
with a complete disclosure and description of how the compounds, compositions,
articles,
devices and/or methods claimed herein are made and evaluated, and are intended
to be purely
exemplary and are not intended to limit the disclosure. Efforts have been made
to ensure
accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some
errors and
deviations should be accounted for. Unless indicated otherwise, parts are
parts by weight,
temperature is in C or is at ambient temperature, and pressure is at or near
atmospheric.
Example 1: Ultra-structural demonstration of a glycoprotein nap surrounding
the
exosporium
To the buffer-washed spore pellets, one milliter (ml) of a 25% glutaraldehyde,
0.1 M
sodium cacodylate solution is supplemented with ruthenium red (1 mg/nil) and
incubated for one
hr at 37 C. Each pellet will is washed in sodium phosphate buffer and fixed
for 3 hr at room
temp. in 2% osmiuni tetroxide in 0.1 M sodium cacodylate solution containing
ratlienium red. A
negative control is treated identically, but rutlieniurn red was omitted from
these two steps.
Spores can be washed in buffer and embedded in 3% agar. Dehydration involves
sequential
treatment with 25%, 50%, 75%, 95%, and 100% ethanol. Afterwards, cells may be
placed
sequentially in propylene oxide, propylene oxide/polybed 812, and pure polybed
812.
Polymerization is carried out at 60 C. Then sections are cut and stained with
a 2% uranyl acetate
solution for 40 min at 37 C, followed by Hanaichi lead citrate for 2 min.
Spores are observed by
transmission electron microscopy.
For ultra-structural observation of B. anthracis spores, upon staining witli
uranyl acetate
and osmium tetroxide, the external basement menibrane of the exosporium inay
be readily visible
separated from the underlying coat layers. After additional ruthenium red
staining, the external
nap is readily demonstrable. It will be demonstrated, using imrnuno-gold
labeling that the
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peptide portion of Bc1A is expressed on the exosporium surface. Furthemiore,
exosporium nap
additionally is rich in carbohydrate. The standard procedures to purify spores
involve renografin
gradients
Example 2: Analysis of glycoproteins, proteins, lipids, and phospholipids
using gel
electrophoresis, glycoprotein staining and matrix assisted-time-of-flight mass
spectrometry
(MALDI-TOF MS)
B. anthracis spores (50 mg wet weight) were extracted witli a urea buffer (50
mM Tris-
HCl, pH 10, 8 M urea, 2% 2=mercaptoethanol) for 15 min at 90 C. The extracted
spores were
centrifuged at 13,000 g for 10 min at room temp. The supernatant was removed
and stored for
protein analysis. Spore protein extract was combined with loading buffer
(35:1) and loaded onto
IPG strips (pH 3-10) using the multiphor II electrophoresis system or other
appropriate piece of
equipment. Next, the strips are rehydrated for focusing at 23,000 Vh for 24
hours. Then, the
strips were equilibrated immediately in SDS equilibrium buffer (50mM Tris-HCI,
pH 8.8, 6M
Urea, 30% (v/v) glycerol, 2% (w/v) SDS, bromophenol blue, trace) for 15
minutes at room
temperature. Afterwards the strips were equilibrated in a second solution of
DTT (10 mg/mL; 65
mM) for 15 minutes at room temperature. The equilibrated strips were loaded on
to a 4-15%
gradient polyacrylamide gels and electrophoresed in Tris-glycine-SDS buffer.
The gels are
stained with ProtoBlue safe with identify protein spots.
To perform the electro-elution, the gel spots are cut out with a scalpel and
destained in
water or another appropriate destaining buffer. Next, the gel slices are
placed in sample tubes
(Millipore) and placed in a electro-eluter (Millipore) with the appropriate
molecular weight cut
off filter. For example, EA1 runs on a gel at approximately 1001cDa so a 100
kDa molecular
weight filter would be used to capture the protein and still allow the
degassed Tris-glycine buffer
to run througli. The protein samples are electro-eluted at 100 Vh for 22-24
hours depending upon
the specific protein being electro-eluted (smaller proteins require less
time). Finally, the protein
samples are washed in their filter with ddHZO three times and centrifuged at
5,000 rpm for 5
minute intervals until the desired volume is reached.
The proteins were then treated with Zip tips (Michron BioResources, Aubuni,
CA) to
remove the SDS and tris-glycine from the glycoprotein solution. Next, an
appropriate enzyme at
the appropriate conditions is used to break apart the protein or chew off the
carbohydrate
component of a glycoprotein. For example, EA1 can be digested using Trypsin
for 3 hours at
room temperature. Next, the samples are Zip Tiped again to remove any salt or
detergent
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contamination; SDS interferes with MALDI ionization and crystallization while
high
concentrations of Tris and glycine in the MALDI preparation interfere with
absorbance of laser
energy by the matrix. The purified samples were mixed with the MALDI matrix
(1:1 v/v solution
of a-cyaimo hydroxycinnamic acid (20 mg/ml in 7:3 v/v acetonitrile: 0.1 %
trifuoroacetic acid)
and 2,5-dihydroxy benzoic acid (20 mg/ml in 7:3 v/v acetonitrile:5% formic
acid), (31). The
molecular weight (MW) of the intact protein will be determined using a Applied
Biosystems
4700 Protein Analyzer MALDI TOF mass spectrometer (Applied Biosystems, Foster
City, CA)
equipped with a 20 Hz nitrogen laser and a reflectron.
For exaniple, EA1 was identified by MALDI TOF MS analysis and can be seen as
an
intensely stained band,<1 00 kDa band, on gel electrophoresis, See figure 3.
There are at least 7
other visible proteins that appeared after staining and will be analyzed by
MALDI TOF MS.
Using MS analysis the following masses were recorded, 983.4373, 1014.571,
1029.5479,
1140.5757, 1179.5699, 1206.5680, 1223.5785, 1228.7073, 1277.6838, 1356.8062,
1359.7783,
1405.7643, 1414.8136, 1424.7617, 1515.8846, 1517.7678, 1526.8829, 1533.7843,
1684.8827,
1709.8922, 1765.9010, 1771.8489, 1857.8329, 1878.9424, 1901.8921, 1934.9288,
1996.9645,
2063.0415, 2230.1863, and 2497.2002 for the gel band corresponding to the <100
kDa band.
Imputing these values into Protein Prospector and searching the entire Swiss-
Prot database for all
species a MOWSE Score of 7.39 x 1014 was obtained for P94217, which
corresponds to S-layer
protein EAI precursor for B. anthracis.
With a MOWSE Score this high the probability that this is any other protein is
almost zero.
Additionally, 46.1 % coverage of the protein was achieved with a mean ppm
error of only 6.3.
Furthermore, MS/MS spectra were taken of each mass above to further support
the sequence of
each peptide analyzed.
Example 3: Lysed spores, Gel electrophoresis, and Electro-elution to isolated
specific
proteins, glycoprotein, oligosaccarides, lipids, or phospholipids
B. anth.racis spores (50 mg wet weight) were extracted with a urea buffer (50
mM Tris-
HC1, pH 10, 8 M urea, 2% 2-mercaptoethanol) for 15 min at 90 C. The extracted
spores were
centrifitged at 13,000 g for 10 min at room temp. The supernatant was removed
and stored for
protein analysis. 35 :1 of spore protein extract was coxnbined with loading
buffer and loaded onto
IPG strips (pH 3-10) using the multiphor II electrophoresis system or other
appropriate piece of
equipment. Next, the strips are rehydrated for focusing at 23,000 Vh for 24
hours. Then, the
strips were equilibrated immediately in SDS equilibrium buffer (50mM Tris-HCI,
pH 8.8, 6M
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Urea, 30% (v/v) glycerol, 2% (w/v) SDS, bromophenol blue, trace) for 15
minutes at room
temperature. Afterwards the strips were equilibrated in a second solution of
DTT (10 mg/mL; 65
mM) for 15 minutes at room temperature. The equilibrated strips were loaded on
to a 4-15%
gradient polyacrylamide gels and electrophoresed in Tris-glycine-SDS buffer.
The gels are
stained with ProtoBlue safe with identify protein spots.
To perform the electro-elution, the gel spots are cut out with a scalpel and
destained in
water or another appropriate destaining buffer. Next, the gel slices are
placed in sample tubes
(Millipore) and placed in a electro-eluter (Millipore) with the appropriate
molecular weight cut
off filter. For example, EA1 runs on a gel at approximately 1001cDa so a 100
kDa molecular
weight filter would be used to capture the protein and still allow the
degassed Tris-glycine buffer
to run through. The protein samples are electro-eluted at 100 Vh for 22-24
hours depending upon
the specific protein being electro-eluted (smaller proteins require less
time). Finally, the protein
samples are washed in their filter with ddH2O three times and centrifuged at
5,000 rpm for 5
minute intervals until the desired volume is reached. Verification of a
successful electro-elution
can be done by re-running the electro-eluted sample on a one dimensional gel
electrophoresis
mini-gel system.
Example 4: Lectin purification of glycoprotein complexes after anthrax spores
have been
lysed
The glycoproteins on the exosporium of the anthrax spore fonn coinplexes with
other
protein, glycoproteins, oligosaccarides, lipids, or phospholipids and can be
isolated by first lysing
the spores by urea extraction buffer or anther lysis method then purify the
complexes by lectins.
The lectins bind to sugars and should therefore bind to BcIA of the exosporium
of the B.
anthracis spore. The Bc1A is also bound to other substances that should stay
attached to it when it
is bound to the lectin. The glycoprotein complexes can then be unbound to the
lectin by washing
the lectin with sugars that it can bind to stronger than the glycoproteins
therefore the sugars will
out compete the glycoproteins for binding space on the lectin leaving a
mixture of glycoprotein
complexes and sugar that did not bind to the lectin. The sugar can be washed
away with a low
molecular weight cut off filter leaving the purified glycoprotein coniplexes.
Potential lectins that
could be used for this procedure include but are not limited to SBA (E-Y
laboratories), APA (E-
Y laboratories), GSA-1 (E-Y laboratories), RCA-I (E-Y laboratories), RCA-II (E-
Y laboratories),
the L-rhamnose-binding lectins STL1, STL2, and STL3 (Tateno et al., 1998).
These lectins can
come in many forms such as but not limited to a gel or on a bead. Using
Anthrax as a novel
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system tlierer are many other microorgansims that may be purified using lectin
technology (Table
1).
Example 5: Size exclusion chromatography
Lysed spores can be ran through a size exclusion column such as, but not
limited to, a
sephacyl column. In this technique, substances with a molecular weight that is
within the range of
the column will be trapped inside the column but any substance outside of the
mass range will go
through the column therefore sorting the substance by size.
Example 6: Spore carbohydrate complexes: antigenic determinants provide
immunity
against infection in a guinea pig model.
The B. anthracis spore, like those of its closely related species, appear to
contain a
carbohydrate component. It has also been shown that a complete immunity to
anthrax requires a
spore component to the vaccine, in addition to protective antigen .
(a) Protection against anthrax infection with lectin purified glycoprotein
complexes
and their antibody response
Groups of five guinea pigs (half male and half female) and groups of three
rabbits (half
male and half female) wil be immunized intramuscularly witli 100 l to 2 mL
volumes of the
following 1) the animal current animal vaccine from Colorado Serum Co.
(positive control); 2)
an adjuvant only plus PBS (negative control); 3) lectin purified glycoprotein
complexes with an
adjuvant. Booster immunizations will be given at 4, 5, 6, 7, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18,
19 or 20 weeks. The animals will be bled via the Saphenous vein or anther
bleeding method at
two and four weelcs and tested for antibody response by an ELISA procedure.
The guinea pigs
will be challenged intramuscularly at week 20 with 100 time LD50 Bacillus
anthracis Ames or
anther strain. The rabbits will be challenged inhalationally at week 20 with
100 time LD50
Bacillus anthracis Vollum, Ames or anther strain or Bacillus cereus G9241 or
another strain that
can cause an anthrax lilce infection. Spore preparations diluted in PBS will
be applied to
Maxisorp ELISA plates. After overnight incubation at 4 C, the coated wells
will be washed with
wash buffer (PBS [pH 7.4], 0.1 % Tween 20, 0.001 % thimerosal). The plates
will then be reacted
with dilutions of the rabbit or guinea pig antiserum. Dilutions will be made
in ELISA dilution
buffer (PBS [pH 7.4], 5% dry skim milk, 0.001% thiinerosal). The secondary
antibody will be
goat anti-rabbit horseradish peroxidase conjugate. Plates will be incubated at
37 C for 1 hr and
then washed six times with wash buffer. The substrate, 2,2'-azinobis (3-
etliylbenzthiazolinesulfonic acid) will be added and the plates will be read
at 405 nnz after
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incubation at room temperature for 15 minutes with a microtiter plate reader
(Dynex). The
ELISA procedure will also be utilized to determine if reactivity exists
against vegetative cells of
A Sterne- 1, Sterne 34F2, or any other suitable strain from anthrax. If such
activity is found, it will
be removed by an absorption procedure. Vegetative cells of A Sterne-1, Sterne
34F2, or other
suitable strain from anthrax will repeatedly be subcultured- to eliminate
spores from the
population and then grown in nutrient broth to mid-logaritlunic phase,
harvested by
centrifugation, washed in PBS, fixed in formalin, and washed extensively in
PBS. The fixed cells
will be added to an aliquot of the antiserum and antibodies against vegetative
cell antigens
allowed to bind at 4 C. The bacteria and the bound antibodies will then be
removed from the
serum by centrifugation. This will be repeated until no vegetative cell
reactivity is detected by
ELISA. Antibodies from the antisera will be purified using a protein A-agarose
affinity column
(Pierce Chemical Co.). Western blot analysis will be carried out to detennine
if an antibody
response to the exosporium glycoprotein complexes occurs and antigenic
epitopes defined.
This protocol will determine if lectin purified glycoprotein spore complexes
can provide
protection against Ames strain of B. antlaracis both cutaneously and
inhalationly. Furthermore,
this experiment expresses the individual antigens within the glycoprotein
complex that are
immunogenic and what types of antibodies are formed to these glycoprotein
complexes.
(b) Protection against several strains of anthrax and other anthrax like
infections
Groups of ten guinea pigs (half male and half female) and groups of six
rabbits (halfmale
and half female) will be immunized intradermally with 100 l to 2 mL volumes
of the following
1) the current animal vaccine made by Colorado Serum Co. (positive control);
2) an adjuvant
only plus PBS (negative control); 3) lectin purified glycoprotein complexes
with an adjuvant.
Booster immunizations can be given at 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20
weeks. The animals can be bled via Saphenous vein or anther bleeding method at
two and four
weeks and tested for antibody response by an ELISA procedure. The guinea pigs
will be broken
up iiito three sub groups in each of the above groups and challenged
cutaneously at week 20 with
100 time LD50 Bacillus anthracis 1)Vollum or other anthrax strain, 2)Ames or
anotlier strain or
3) Bacillus cereus G9241 or another strain that can cause an anthrax lilce
infection. The rabbits
will be brolcen up into three sub groups within each group and challenged
inhalationly at weelc 20
wit11100 time LD50 Bacillus antlaf acis 1) Vollum or other anthrax strain, 2)
Ames or anther strain
or 3) Bacillus cereus G9241 or another strain that can cause an anthrax lilce
infection. The above
protocol will determine if lectin purified glycoprotein spore complexes will
provide protection
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against B. anthracis and other bacteria that cause anthrax lilce infections
both cutaneously and
inhalationally.
Example 7 One Dimensional Gel of Lectin Purified Complexes From B. anthracis
FIG. 3 is a one-dimensional SDS gel that contains both urea extracted spores
and lectin
purified complexes. Sterne 34F2 spores were obtained from Colorado Seruin Co.
The spores
were grown on nutrient agar plates (Difco, Detroit, MI) for one week when
sporulation was
complete for most of the bacterium (>95%). The spores were harvest from the
plates using
milliQ water set to 18.2 milli0hms. The spores were frozen at -80 degrees C
overnight. The
next day, the spores were allowed to thaw at room temperature to lyse any of
the remaining
vegetative cells (approximately 3 hours). Next, the spores were washed
centrifuging at 10,000
rpm for 10 minutes at 4 degrees C. The water on top of the spores was decanted
off and new
water was added on top to wash the spores. The amount of water added was equal
to the volume
of spores in the tube. The tube was vortexed and spun again 10,000 rpm for 10
minutes at 4
degrees C. The wash procedure just described was repeated three times until
the water on the top
of the spores was clear. The final volume of water added was equal to the
volume of centrifuged
spores in the tube. The spores were counted an analyzed for purity using phase
contrast
microscopy. Next, the spores were urea extracted. For urea extracted spores
1000 uL of
concentrated B. anthracis suspension (1.27 x 10117 spores per microliter at
99.76% pure spore)
was centrifuged at 10,000 rpm for 10 minutes. Then, the liquid on top was
decanted off. Next,
300 microliters of urea extract buffer (50 mM Tris-HCl, ph 10, 8 M urea, 2% 2-
mercaptoethanol)
(Fisher Scientific) was added to the spores and vortexed until all the spores
were dissolved in the
solution. The urea solution was heated to 90 degrees C for 15 minutes. Then,
the urea extracted
spores were centrifuged at 10,000 rpm for 10 minutes. The supernatant was
removed and the
particulate at the bottom was thrown away. Half the supematant was used in the
urea extracted
lanes of the gel shown in this figure. The other half of the supernatant was
used for lectin
purification. Two mL of SBA lectin bound to agrose beads was placed in a
gravity column
(Fisher Scientific). The SBA lectin was washed using 4 mL of water. Next, 150
microliters of
urea extracted spores was placed on the coluinn and allowed to sit for 1 hour.
Then, the excess
unbound material was allowed to drain off into a waste container. Next, 1.2 mL
of 0.1M D-
galactose was added to the column and allowed to sit for 1 hour. Then, the
column was allowed
to drain and small samples of the bound material were collected (about 300
microliters). The
bound samples were then run on an SDS page gel described below. The urea
extracted spores
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(the supernatant) or lectin treated urea extracted spores was added to twice
the volume of sample
buffer (50 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 10 %
glycerol, 5% 2-
mercaptoethanol, 0.02% bromophenol blue) (Fisher Scientific) and heated to 95
degrees C for 4
minutes. Fifteen microliters of a kaleidoscope Prestained Standard (BioRad)
was used in one
lane. The prestained standard was, also, heated at 95 degrees C for 4 minutes
prior to being
loaded onto the gel. Fifteen microliters of the urea extracted spores plus
sample buffer or 15
microliters of lectin treated urea extracted spores plus sample buffer was
loaded on to a 4-15%
polyacrylamide minigel system (BioRad). The sample was electrophoresed using
Tris-Glycine-
SDS Buffer (Fisher Scientific). The gel was ran at 100V for 2 hours. The gel
was washed three
times with milliQ water set to 18.2 milliOhms for 15 minutes three times
before staining. The
gel was stained using gel code blue comassee stain overnight (Pierce,
Rockford, IL). Finally, the
gel was washed three times for 15 minutes to remove any excess stain. Lanes A,
C, and E are all
urea extracted spores. Lane B is the lectin isolated urea extracted spores.
There are 7 bands in
this lane. One band contains EA1. Lane D is the kaleidoscope prestained
standard.
Example 8: Urea Extracted Spores Before Lectin Treatment
FIG. 4 shows urea extracted spores before lectin treatment. Sterne 34F2 spores
were
obtained from Colorado Serum Co. The spores were grown on nutrient agar plates
(Difco,
Detroit, MI) for one weelc when sporulation was complete for most of the
bacterium (>95%).
The spores were harvest from the plates using milliQ water set to 18.2
milliOhms. The spores
were frozen at -80 degrees C overnight. The next day, the spores were allowed
to thaw at room
temperature to lyse any of the remaining vegetative cells (approximately 3
hours). Next, the
spores were washed centrifuging at 10,000 rpm for 10 minutes at 4 degrees C.
The water on top
of the spores was decanted off and new water was added on top to wash the
spores. The ainount
of water added was equal to the volume of spores in the tube. The tube was
vortexed and spun
again 10,000 rpm for 10 minutes at 4 degrees C. The wash procedure just
described was repeated
three times until the water on the top of the spores was clear. The final
volume of water added
was equal to the volume of centrifuged spores in the tube. The spores were
counted an analyzed
for purity using phase contrast microscopy. Next, the spores were urea
extracted. For urea
extracted spores 1000 uL of concentrated B. aratlaracis suspension (1.27 x
10~7 spores per
microliter at 99.76% pure spore) was centrifuged at 10,000 rpm for 10 minutes.
Then, the liquid
on top was decanted off. Next, 300 microliters of urea extract buffer (50 mM
Tris-HC1, ph 10, 8
M urea, 2% 2-inercaptoethanol) (Fisher Scientific) was added to the spores and
vortexed until all
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the spores were dissolved in the solution. The urea solution was heated to 90
degrees C for 15
minutes. Then, the urea extracted spores were centrifuged at 10,000 rpm for 10
minutes. The
supematant was removed and the particulate at the bottom was thrown away.
The urea extracted spore protein extract (the supernatant) was combined with
loading
buffer and loaded onto IPG strips (pH 3-10) using the multiphor II
electrophoresis system
(Amersham) or other appropriate piece of equipment. Next, the strips are
rehydrated for focusing
at 23,000 Vh for 24 hours. Then, the strips were equilibrated immediately in
SDS equilibrium
buffer (50mM Tris-HCI, pH 8.8, 6M Urea, 30% (v/v) glycerol, 2% (w/v) SDS,
bromophenol
blue, trace) for 15 minutes at room temperature. Afterwards the strips were
equilibrated in a
second solution of DTT (10 mg/mL; 65 mM) for 15 nlinutes at room teinperature.
The
equilibrated strips were loaded on to a 4-15% gradient polyacrylainide gels
and electrophoresed
in Tris-glycine-SDS buffer. The gel was stained for glycoproteins with ECL
glycoprotein
detection system (Amersham Biosciences) according to the manufacturer's
description. The urea
extracted spores reveal two glycoproteins.
Example 9: MALDI TOF MS Spectrum of an Anthrax Glycoprotein
FIG. 5 show a matrix-assisted laser desorption/ionization (MALDI) time-of-
flight (TOF)
mass spectrum of a gel slice obtained from a one diinensional gel, which is
shown in Figure 3.
The protein was identified as B. azzthz acis S-layer protein EA 1 pre-cursor
(EAl ID) from Swiss-
Prot database, P94217, and with a MOWSE score of 7.39 x 10+14. With a score
this high the
probability that this is any other protein is almost zero. Additionally, 46.1
% coverage of the
protein was achieved with a mean ppm error of only 6.3. All of the masses
above a signal-to-
noise threshold of 10:1 were applied to data analyze, which generated the
above identification.
The MADLI TOF MS used in this experiment was a Applied Biosystems 4700 Protein
Identification system. To generate this spectrum the following protocol was
employed. After
staining of the gel several spots of interest were selected for MS analysis.
These spots were
excised using a cleaned autoclaved razor blade and added to a 1.5 n7L
centrifuge tube. The gel
slices were then de-stained for 45 min with 200 uL of 100 mM solution of
ammonium
bicarbonate in 50 % acetonitrile. The tubes are then vacuum dried at 37 C
until they are dry.
Next, the samples are reduced by adding 100 uL of 2 mM TCEP (Tris (2-
carboxyethyl)phosphine, in 25 nM ammonium bicarbonate (pH 8.0) and allowed to
incubate for
15 minutes at 37 C with slight agitation. The supernant is removed and 100 uL
of 20 mM
iodoacetanlide in 25 mM ammonium bicarbonate (pH8.0) is added and allowed to
sit in the darlc
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fro 15 minutes. The gels are then washed three times with 200 uL of 25 mM
ammonium
bicarbonate for 15 minutes, then dried with vacuum centrifugation. The gels
are re-hydrated with
20 uL of 0.02 ug/uL of sequencing grade modified trypsin in 10 % acetonitrile,
with 40 mM
ammonium bicarbonate (pH 8.0) and 0.1 % n-octylgucoside for one hour at room
temperature.
Next, 50 uL of 10 % acetonitrile with 40 mM ammonium bicarbonate )pH 8.0) is
added to the
tubes and allowed to sit for 5 ininutes. The supernant is removed placed into
a fresh 1.5 mL
centrifuge tube and vacuum centrifuged to dryness. Next, 200 uL of pure water
is added and then
spun to dryness again. This is repeated three times. Finally, on the forth re-
suspension the
solution is dried until only 10 uL of sample remains. This remaining solution
is then ready for
MALDI TOF MS analysis. For MS analysis 1 uL of sample is mixed with 1 uL of
matrix and
spotted until the stainless steel probe for analysis. The matrix used is 2, 5
di-hydroxybenzoic
acid (DHB) in 80/20 methanol water matrix with a saturated solution of DHB.
After the spot
dries the sample is running using a standard conditions with an Applied
Biosystems 4700 Protein
Analyzer MS.
Example 10: Protein and Nucleic Acid Sequences for PCT Application
B. anthracis BcIA (40048) - (Q52NY8)
2. SQ SEQUENCE 322 AA; 30133 MW; B036C1F1F4432E02 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGPTGPTGPT GPTGPTGPTG PTGPTGDTGT
TGPTGPTGPT GPTGPTGDTG TTGPTGPTGP TGPTGPTGPT GPTGPTGPTG PTGATGLTGP
TGPTGPSGLG LPAGLYAFNS GGISLDLGIN DPVPFNTVGS QFGTAISQLD ADTFVISETG
FYKITVIANT ATASVLGGLT IQVNGVPVPG TGSSLISLGA PIVIQAITQI TTTPSLVEVI
VTGLGLSLAL GTSASIIIEK VA
1. SQ Sequence 969 BP; 265 A; 247 C; 231 G; 226 T; 0 other; 3713744812 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccaactggac caactgggcc gactgggcca actggaccaa ctgggccgac tgggccaact 300
ggaccaactg ggccgactgg gccaactgga ccaactgggc caactggaga cactggtact 360
actggaccaa ctgggccaac tggaccaact ggaccaactg ggccaactgg agacactggt 420
actactggac caactgggcc aactggacca actggaccaa ctgggccgac tggaccgact 480
gggccgactg ggccaactgg gccaactggg ccaactggtg ctaccggact gactggaccg 540
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actggaccga ctgggccatc cggactagga cttccagcag gactatatgc atttaactcc 600
ggtgggattt ctttagattt aggaattaat gatccagtac catttaatac tgttggatct 660
cagtttggta cagcaatttc tcaattagat gctgatactt tcgtaattag tgaaactgga 720
ttctataaaa ttactgttat cgctaatact gcaacagcaa gtgtattagg aggtcttaca 780
atccaagtga atggagtacc tgtaccaggt actggatcaa gtttgatttc actcggagca 840
cctatcgtta ttcaagcaat tacgcaaatt acgacaactc catcattagt tgaagtaatt 900
gttacagggc ttggactatc actagctctt ggcacga~Itg catccattat tattgaaaaa 960
gttgcttaa 969
B. anthracis BcIA (A16R) - (Q52NZO)
4. SQ SEQUENCE 388 AA; 35793 MW; 50767CAB307A5A7F CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGDTGTTGPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGPTGPTG PTGPTGDTGT TGPTGPTGPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGDTGTTGPT GPTGPTGPTG PTGPTGPTGA
TGLTGPTGPT GPSGLGLPAG LYAFNSGGIS LDLGINDPVP FNTVGSQFGT AISQLDADTF
VISETGFYKI TVIANTATAS VLGGLTIQVN GVPVPGTGSS LISLGAPIVI QAITQITTTP
SLVEVIVTGL GLSLALGTSA SIIIEKVA
3. SQ Sequence 1167 BP; 321 A; 309 C; 285 G; 252 T; 0 other; 3217654551 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccgactgggc caactggacc aactgggcca actggagaca ctggtactac tggaccaact 300
gggccaactg gaccaactgg gccgactggg ccaactggac caactgggcc gactgggcca 360
actggaccaa ctgggccaac tggaccaact ggaccaactg ggccaactgg accaactgga 420
ccaactgggc caactggaga cactggtact actggaccaa ctgggccaac tggaccaact 480
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 540
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 600
ccaactggac caactggacc aactgggcca actggagaca ctggtactac tggaccaact 660
gggccaactg gaccaactgg accaactggg ccaactggac caactgggcc aactggtgct 720
accggactga ctggaccgac tggaccgact gggccatccg gactaggact tccagcagga 780
ctatatgcat ttaactccgg tgggatttct ttagatttag gaattaatga tccagtacca 840
tttaatactg ttggatctca gtttggtaca gcaatttctc aattagatgc tgatactttc 900
gtaattagtg aaactggatt ctataaaatt actgttatcg ctaatactgc aacagcaagt 960
gtattaggag gtcttacaat ccaagtgaat ggagtacctg taccaggtac tggatcaagt 1020
ttgatttcac tcggagcacc tatcgttatt caagcaatta cgcaaattac gacaactcca 1080
tcattagttg aagtaattgt tacagggctt ggactatcac tagctcttgg cacgagtgca 1140
tccattatta ttgaaaaagt tgcttaa 1167
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B. anthracis BcIA (CIPA2) - (Q83TLO)
6. SQ SEQUENCE 262 AA; 25006 MW; CB03E1E413646488 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGDTGTTGPT GPTGPTGPTG PTGATGLTGP
TGPTGPSGLG LPAGLYAFNS GGISLDLGIN DPVPFNTVGS QFGTAISQLD ADTFVISETG
FYKITVIANT ATASVLGGLT IQVNGVPVPG TGSSLISLGA PIVIQAITQI TTTPSLVEVI
VTGLGLSLAL GTSASIIIEK VA '
5. SQ Sequence 789 BP; 223 A; 189 C; 173 G; 204 T; 0 other; 668699339 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccaactggac caactgggcc aactgggcca actggagaca ctggtactac tggaccaact 300
gggccaactg gaccaactgg accaactggg ccaactggtg ctaccggact gactggaccg 360
actggaccga ctgggccatc cggactagga cttccagcag gactatatgc atttaactcc 420
ggtgggattt ctttagattt aggaattaat gatccagtac catttaatac tgttggatct 480
cagtttggta cagcaatttc tcaattagat gctgatactt tcgtaattag tgaaactgga 540
ttctataaaa ttactgttat cgctaatact gcaacagcaa gtgtattagg aggtcttaca 600
atccaagtga atggagtacc tgtaccaggt actggatcaa gtttgatttc actcggagca 660
cctatcgtta ttcaagcaat tacgcaaatt acgacaactc catcattagt tgaagtaatt 720
gttacagggc ttggactatc actagctctt ggcacgagtg catccattat tattgaaaaa 780
gttgcttaa 789
B. anthracis Bc1A (7611) - (Q83UV2)
8. SQ SEQUENCE 253 AA; 24218 MW; 10231F93AD9A1385 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGPTGPTG PTGDTGTTGP TGPTGPTGPT GPTGATGLTG PTGPTGPSGL
GLPAGLYAFN SGGISLDLGI NDPVPFNTVG SQFGTAISQL DADTFVISET GFYKITVIAN
TATASVLGGL TIQVNGVPVP GTGSSLISLG APIVIQAITQ ITTTPSLVEV IVTGLGLSLA
LGTSASIIIE KVA
7. SQ Sequence 762 BP; 216 A; 182 C; 165 G; 199 T; 0 other; 3124681291 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tggaccaact gggccaactg gaccaactgg gccaactggg 240
ccaactggag acactggtac tactggacca actgggccaa ctggaccaac tggaccaact 300
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gggccaactg gtgctaccgg actgactgga ccgactggac cgactgggcc atccggacta 360
ggacttccag caggactata tgcatttaac tccggtggga tttctttaga tttaggaatt 420
aatgatccag taccatttaa tactgttgga tctcagtttg gtacagcaat ttctcaatta 480
gatgctgata ctttcgtaat tagtgaaact ggattctata aaattactgt tatcgctaat 540
actgcaacag caagtgtatt aggaggtctt acaatccaag tgaatggagt acctgtacca 600
ggtactggat caagtttgat ttcactcgga gcacctatcg ttattcaagc aattacgcaa 660
attacgacaa ctccatcatt agttgaagta attgttacag ggcttggact atcactagct 720
cttggcacga gtgcatccat tattattgaa aaagttgctt aa 762
B. anthracis BcIA (ATCC4229) - (Q83WA5)
10. SQ SEQUENCE 223 AA; 21665 MW; 450F8ECB33FBC58E CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGDTGT
TGPTGPTGPT GPTGATGLTG PTGPTGPSGL GLPAGLYAFN SGGISLDLGI NDPVPFNTVG
SQFGTAISQL DADTFVISET GFYKITVIAN TATASVLGGL TIQVNGVPVP GTGSSLISLG
APIVIQAITQ ITTTPSLVEV IVTGLGLSLA LGTSASIIIE KVA
9. SQ Sequence 672 BP; 195 A; 152 C; 136 G; 189 T; 0 other; 1857948650 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccaactgg accaactggg ccaactgggc caactggaga cactggtact 180
actggaccaa ctgggccaac tggaccaact gggccaactg gtgctaccgg actgactgga 240
ccgactggac cgactgggcc atccggacta ggacttccag caggactata tgcatttaac 300
tccggtggga tttctttaga tttaggaatt aatgatccag taccatttaa tactgttgga 360
tctcagtttg gtacagcaat ttctcaatta gatgctgata ctttcgtaat tagtgaaact 420
ggattctata aaattactgt tatcgctaat actgcaacag caagtgtatt aggaggtctt 480
acaatccaag tgaatggagt acctgtacca ggtactggat caagtttgat ttcactcgga 540
gcacctatcg ttattcaagc aattacgcaa attacgacaa ctccatcatt agttgaagta 600
attgttacag ggcttggact atcactagct cttggcacga gtgcatccat tattattgaa 660
aaagttgctt aa 672
B. anthracis BcIA (CIP5725) - (Q83WA6)
12. SQ SEQUENCE 244 AA; 23452 MW; AC95F5F306ACD892 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGDTGTTG PTGPTGPTGP TGPTGATGLT GPTGPTGPSG LGLPAGLYAF
NSGGISLDLG INDPVPFNTV GSQFGTAISQ LDADTFVISE TGFYKITVIA NTATASVLGG
LTIQVNGVPV PGTGSSLISL GAPIVIQAIT QITTTPSLVE VIVTGLGLSL ALGTSASIII
EKVA
11. SQ Sequence 735 BP; 210 A; 173 C; 156 G; 196 T; 0 other; 1433959005 CRC32;
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atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactggacca 180
actgggccaa ctggaccaac tgggccaact gggccaactg gagacactgg tactactgga 240
ccaactgggc caactggacc aactggacca actgggccaa ctggtgctac cggactgact 300
ggaccgactg gaccgactgg gccatccgga ctaggacttc cagcaggact atatgcattt 360
aactccggtg ggatttcttt agatttagga attaatgatc cagtaccatt taatactgtt 420
ggatctcagt ttggtacagc aatttctcaa ttagatgctg atactttcgt aattagtgaa 480
actggattct ataaaattac tgttatcgct aatactgcaa cagcaagtgt attaggaggt 540
cttacaatcc aagtgaatgg agtacctgta ccaggtactg gatcaagttt gatttcactc 600
ggagcaccta tcgttattca agcaattacg caaattacga caactccatc attagttgaa 660
gtaattgtta cagggcttgg actatcacta gctcttggca cgagtgcatc cattattatt 720
gaaaaagttg cttaa 735
B. anthracis BcIA (ATCC6602) - (Q83WA7)
14. SQ SEQUENCE 253 AA; 24208 MW; 01293B56EDB92731 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGDTGTTG PTGPTGPTGP TGPTGPTGPT GPTGATGLTG PTGPTGPSGL
GLPAGLYAFN SGGISLDLGI NDPVPFNTVG SQFGTAISQL DADTFVISET GFYKITVIAN
TATASVLGGL TIQVNGVPVP GTGSSLISLG APIVIQAITQ ITTTSSLVEV IVTGLGLSLA
LGTSASIIIE KVA
13. SQ Sequence 762 BP; 216 A; 182 C; 164 G; 200 T; 0 other; 645088734 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tggaccaact gggccaactg gagacactgg tactactgga 240
ccaactgggc caactggacc aactggacca actgggccaa ctggaccaac tggaccaact 300
gggccaactg gtgctaccgg actgactgga ccgactggac cgactgggcc atccggacta 360
ggacttccag caggactata tgcatttaac tccggtggga tttctttaga tttaggaatt 420
aatgatccag taccatttaa tactgttgga tctcagtttg gtacagcaat ttctcaatta 480
gatgctgata ctttcgtaat tagtgaaact ggattctata aaattactgt tatcgctaat 540
actgcaacag caagtgtatt aggaggtctt acaatccaag tgaatggagt acctgtacca 600
ggtactggat caagtttgat ttcactcgga gcacctatcg ttattcaagc aattacgcaa 660
attacgacaa cttcctcatt agttgaagta attgttacag ggcttggact atcactagct 720
cttggcacga gtgcatccat tattattgaa aaagttgctt aa 762
B. anthracis BcIA (CIP53169) - (Q83WA8)
16. SQ SEQUENCE 370 AA; 34262 MW; 064CEDCEFDEBB127 CRC64;
-79-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGPTGPTGPT GPTGPTGDTG TTGPTGPTGP
TGPTGPTGDT GTTGPTGPTG PTGPTGPTGP TGPTGPTGPT GPTGPTGPTG PTGDTGTTGP
TGPTGPTGPT GPTGDTGTTG PTGPTGPTGP TGPTGPTGPT GATGLTGPTG PTGPSGLGLP
AGLYAFNSGG ISLDLGINDP VPFNTVGSQF GTAISQLDAD TFVISETGFY KITVIANTAT
ASVLGGLTIQ VNGVPVPGTG SSLISLGAPI VIQAITQITT TPSLVEVIVT GLGLSLALGT
SASIIIEKVA
15. SQ Sequence 1113 BP; 307 A; 291 C; 269 G; 246 T; 0 other; 2173493146
CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccaactggac caactgggcc gactgggcca actggaccaa ctgggccgac tgggccaact 300
ggaccaactg ggccaactgg agacactggt actactggac caactgggcc aactggacca 360
actggaccaa ctgggccaac tggagacact ggtactactg gaccaactgg gccaactgga 420
ccaactggac caactgggcc gactggaccg actgggccga ctgggccaac tggaccaact 480
gggccgactg ggccaactgg accaactggg ccaactggag acactggtac tactggacca 540
actgggccaa ctggaccaac tggaccaact gggccaactg gagacactgg tactactgga 600
ccaactgggc caactggacc aactggacca actgggccaa ctggaccaac tgggccaact 660
ggtgctaccg gactgactgg accgactgga ccgactgggc catccggact aggacttcca 720
gcaggactat atgcatttaa ctccggtggg atttctttag atttaggaat taatgatcca 780
gtaccattta atactgttgg atctcagttt ggtacagcaa tttctcaatt agatgctgat 840
actttcgtaa ttagtgaaac tggattctat aaaattactg ttatcgctaa tactgcaaca 900
gcaagtgtat taggaggtct tacaatccaa gtgaatggag tacctgtacc aggtactgga 960
tcaagtttga tttcactcgg agcacctatc gttattcaag caattacgca aattacgaca 1020
actccatcat tagttgaagt aattgttaca gggcttggac tatcactagc tcttggcacg 1080
agtgcatcca ttattattga aaaagttgct taa 1113
B. anthracis BcIA (CIP8189) - (Q83WA9)
18. SQ SEQUENCE 391 AA; 36071 MW; E8B7B61480FD9DB9 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGDTGTTGPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGDT GTTGPTGPTG PTGPTGPTGD TGTTGPTGPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGDTGTTG PTGPTGPTGP TGPTGDTGTT GPTGPTGPTG PTGPTGPTGP
TGATGLTGPT GPTGPSGLGL PAGLYAFNSG GISLDLGIND PVPFNTVGSQ FGTAISQLDA
DTFVISETGF YKITVIANTA TASVLGGLTI QVNGVPVPGT GSSLISLGAP IVIQAITQIT
TTPSLVEVIV TGLGLSLALG TSASIIIEKV A
17. SQ Sequence 1176 BP; 323 A; 310 C; 288 G; 255 T; 0 other; 1987561614
CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
-80-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccgactgggc caactggacc aactgggcca actggagaca ctggtactac tggaccaact 300
gggccaactg gaccaactgg gccgactggg ccaactggac caactgggcc gactgggcca 360
actggaccaa ctgggccaac tggagacact ggtactactg gaccaactgg gccaactgga 420
ccaactggac caactgggcc aactggagac actggtacta ctggaccaac tgggccaact 480
ggaccaactg gaccaactgg gccgactgga ccgactgggc cgactgggcc aactggacca 540
actgggccga ctgggccaac tggaccaact gggccaactg gagacactgg tactactgga 600
ccaactgggc caactggacc aactggacca actgggccaa ctggagacac tggtactact 660
ggaccaactg ggccaactgg accaactgga ccaactgggc caactggacc aactgggcca 720
actggtgcta ccggactgac tggaccgact ggaccgactg ggccatccgg actaggactt 780
ccagcaggac tatatgcatt taactccggt gggatttctt tagatttagg aattaatgat 840
ccagtaccat ttaatactgt tggatctcag tttggtacag caatttctca attagatgct 900
gatactttcg taattagtga aactggattc tataaaatta ctgttatcgc taatactgca 960
acagcaagtg tattaggagg tcttacaatc caagtgaatg gagtacctgt accaggtact 1020
ggatcaagtt tgatttcact cggagcacct atcgttattc aagcaattac gcaaattacg 1080
acaactccat cattagttga agtaattgtt acagggcttg gactatcact agctcttggc 1140
acgagtgcat ccattattat tgaaaaagtt gcttaa 1176
B. anthracis BcIA (Sterne CIP7702) -(Q83WB0)
20. SQ SEQUENCE 445 AA; 40709 MW; DAF461B2B6FFA247 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGPTGPTG DTGTTGPTGP TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGPTGDTG TTGPTGPTGP TGPTGPTGDT GTTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GPTGPTGPTG PTGDTGTTGP TGPTGPTGPT GPTGDTGTTG PTGPTGPTGP
TGPTGDTGTT GPTGPTGPTG PTGPTGDTGT TGPTGPTGPT GPTGPTGPTG PTGPTGATGL
TGPTGPTGPS GLGLPAGLYA FNSGGISLDL GINDPVPFNT VGSQFGTAIS QLDADTFVIS
ETGFYKITVI ANTATASVLG GLTIQVNGVP VPGTGSSLIS LGAPIVIQAI TQITTTPSLV
EVIVTGLGLS LALGTSASII IEKVA
19. SQ Sequence 1338 BP; 368 A; 360 C; 333 G; 277 T; 0 other; 688694428 CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccgact gggccaactg gaccaactgg gccaactgga 240
gacactggta ctactggacc aactgggccg actgggccaa ctggaccaac tgggccaact 300
ggagacactg gtactactgg accaactggg ccaactggac caactgggcc gactgggcca 360
actggaccaa ctgggccgac tgggccaact ggaccaactg ggccaactgg agacactggt 420
actactggac caactgggcc aactggacca actggaccaa ctgggccaac tggagacact 480
ggtactactg gaccaactgg gccaactgga ccaactggac caactgggcc gactggaccg 540
actgggccga ctgggccaac tggaccaact gggccgactg ggccaactgg accaactggg 600
-81-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
ccaactggag acactggtac tactggacca actgggccaa ctggaccaac tggaccaact 660
gggccaactg gagacactgg tactactgga ccaactgggc caactggacc aactggacca 720
actgggccaa ctggagacac tggtactact ggaccaactg ggccaactgg accaactgga 780
ccaactgggc caactggaga cactggtact actggaccaa ctgggccaac tggaccaact 840
ggaccaactg ggccaactgg accaactgga ccaactgggc caactggtgc taccggactg 900
actggaccga ctggaccgac tgggccatcc ggactaggac ttccagcagg actatatgca 960
tttaactccg gtgggatttc tttagattta ggaattaatg atccagtacc atttaatact 1020
gttggatctc agtttggtac agcaatttct caattagatg ctgatacttt cgtaattagt 1080
gaaactggat tctataaaat tactgttatc gctaatactg caacagcaag tgtattagga 1140
ggtcttacaa tccaagtgaa tggagtacct gtaccaggta ctggatcaag tttgatttca 1200
ctcggagcac ctatcgttat tcaagcaatt acgcaaatta cgacaactcc atcattagtt 1260
gaagtaattg ttacagggct tggactatca ctagctcttg gcacgagtgc atccattatt 1320
attgaaaaag ttgcttaa 1338
B. anthracis BcIA (Ames) - (Q81JD7, Q6KVS0, Q7BYA5)
22. SQ SEQUENCE 382 AA; 35305 MW; 1DB4ED430DA07037 CRC64;
MSNNNYSNGL NPDESLSASA FDPNLVGPTL PPIPPFTLPT GPTGPTGPTG PTGPTGPTGP
TGPTGPTGPT GDTGTTGPTG PTGPTGPTGP TGDTGTTGPT GPTGPTGPTG PTGPTGPTGD
TGTTGPTGPT GPTGPTGPTG DTGTTGPTGP TGPTGPTGPT GPTGPTGPTG PTGPTGPTGP
TGPTGDTGTT GPTGPTGPTG PTGPTGDTGT TGPTGPTGPT GPTGPTGPTG PTGATGLTGP
TGPTGPSGLG LPAGLYAFNS GGISLDLGIN DPVPFNTVGS QFGTAISQLD ADTFVISETG
FYKITVIANT ATASVLGGLT IQVNGVPVPG TGSSLISLGA PIVIQAITQI TTTPSLVEVI
VTGLGLSLAL GTSASIIIEK VA
21. SQ Sequence 1149 BP; 317 A; 301 C; 279 G; 252 T; 0 other; 3918642356
CRC32;
atgtcaaata ataattattc aaatggatta aaccccgatg aatctttatc agctagtgca 60
tttgacccta atcttgtagg acctacatta ccaccgatac caccatttac ccttcctacc 120
ggaccaactg ggccgactgg accgactggg ccgactgggc caactggacc aactgggccg 180
actgggccaa ctggaccaac tgggccaact ggagacactg gtactactgg accaactggg 240
ccgactgggc caactggacc aactgggcca actggagaca ctggtactac tggaccaact 300
gggccaactg gaccaactgg gccgactggg ccaactggac caactgggcc aactggagac 360
actggtacta ctggaccaac tgggccaact ggaccaactg gaccaactgg gccaactgga 420
gacactggta ctactggacc aactgggcca actggaccaa ctggaccaac tgggccgact 480
ggaccgactg ggccgactgg gccaactgga ccaactgggc cgactgggcc aactggacca 540
actgggccaa ctggagacac tggtactact ggaccaactg ggccaactgg accaactgga 600
ccaactgggc caactggaga cactggtact actggaccaa ctgggccaac tggaccaact 660
ggaccaactg ggccaactgg accaactggg ccaactggtg ctaccggact gactggaccg 720
actggaccga ctgggccatc cggactagga cttccagcag gactatatgc atttaactcc 780
ggtgggattt ctttagattt aggaattaat gatccagtac catttaatac tgttggatct 840
cagtttggta cagcaatttc tcaattagat gctgatactt tcgtaattag tgaaactgga 900
ttctataaaa ttactgttat cgctaatact gcaacagcaa gtgtattagg aggtcttaca 960
-82-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
atccaagtga atggagtacc tgtaccaggt actggatcaa gtttgatttc actcggagca 1020
cctatcgtta ttcaagcaat tacgcaaatt acgacaactc catcattagt tgaagtaatt 1080
gttacagggc ttggactatc actagctctt ggcacgagtg catccattat tattgaaaaa 1140
gttgcttaa 1149
B. anthracis EA1- (P94217, Q612R2, Q6KWJ3)
24. SQ SEQUENCE 862 AA; 91362 MW; CB16B202F62CCCA0 CRC64;
MAKTNSYKKV IAGTMTAAMV AGIVSPVAAA GKSFPDVPAG HWAEGSINYL VDKGAITGKP
DGTYGPTESI DRASAAVIFT KILNLPVDEN AQPSFKDAKN IWSSKYIAAV EKAGVVKGDG
KENFYPEGKI DRASFASMLV SAYNLKDKVN GELVTTFEDL LDHWGEEKAN ILINLGISVG
TGGKWEPNKS VSRAEAAQFI ALTDKKYGKK DNAQAYVTDV KVSEPTKLTL TGTGLDKLSA
DDVTLEGDKA VAIEASTDGT SAVVTLGGKV APNKDLTVKV KNQSFVTKFV YEVKKLAVEK
LTFDDDRAGQ AIAFKLNDEK GNADVEYLNL ANHDVKFVAN NLDGSPANIF EGGEATSTTG
KLAVGIKQGD YKVEVQVTKR GGLTVSNTGI ITVKNLDTPA SAIKNVVFAL DADNDGVVNY
GSKLSGKDFA LNSQNLVVGE KASLNKLVAT IAGEDKVVDP GSISIKSSNH GIISVVNNYI
TAEAAGEATL TIKVGDVTKD VKFKVTTDSR KLVSVKANPD KLQVVQNKTL PVTFVTTDQY
GDPFGANTAA IKEVLPKTGV VAEGGLDVVT TDSGSIGTKT IGVTGNDVGE GTVHFQNGNG
ATLGSLYVNV TEGNVAFKNF ELVSKVGQYG QSPDTKLDLN VSTTVEYQLS KYTSDRVYSD
PENLEGYEVE SKNLAVADAK IVGNKVVVTG KTPGKVDIHL TKNGATAGKA TVEIVQETIA
IKSVNFKPVQ TENFVEKKIN IGTVLELEKS NLDDIVKGIN LTKETQHKVR VVKSGAEQGK
LYLDRNGDAV FNAGDVKLGD VTVSQTSDSA LPNFKADLYD TLTTKYTDKG TLVFKVLKDK
DVITSEIGSQ AVHVNVLNNP NL
23. SQ Sequence 2589 BP; 926 A; 421 C; 515 G; 727 T; 0 other; 2474321808
CRC32;
atggcaaaga ctaactctta caaaaaagta atcgcaggta caatgacagc agcaatggta 60
gcaggtattg tatctccagt agcagcagca ggtaaatcat tcccagacgt tccagctgga 120
cattgggcag aaggttctat taattactta gtagataaag gtgcaattac aggtaagcca 180
gacggtacat atggtccaac cgaatcaatc gatcgtgctt ctgcagctgt aatcttcact 240
aaaattttaa atttaccagt tgatgaaaat gctcagcctt ctttcaaaga tgctaaaaat 300
atttggtctt caaaatatat tgcagcagtt gaaaaagctg gcgttgttaa aggtgatggc 360
aaagaaaact tctatccaga aggaaagatt gaccgtgctt catttgcttc tatgttagta 420
agtgcttata acttaaaaga taaagttaac ggcgagttag ttacgacatt tgaagattta 480
ttagatcatt ggggtgaaga gaaagcaaac atcctaatta accttggaat ctctgtaggt 540
actggtggta aatgggagcc aaataaatct gtatctcgtg cagaagcagc tcaatttatc 600
gcattaacag ataaaaaata tggaaaaaaa gataatgcac aagcgtatgt aactgatgtg 660
aaagtttctg agccaacgaa attaacatta acaggtactg gcttagacaa actttctgct 720
gatgatgtaa ctcttgaagg agacaaagca gttgcaatcg aagcaagtac tgatggtact 780
tctgcagttg taacacttgg tggcaaagta gctccaaata aagaccttac tgtaaaagtg 840
aaaaatcaat cattcgtaac gaaattcgta tacgaagtga aaaaattagc agtagaaaaa 900
cttacatttg atgatgatcg cgctggtcaa gcaattgctt tcaaattaaa cgatgaaaaa 960
ggtaacgctg atgttgagta cttaaactta gcaaaccatg acgtcaaatt tgtagcgaat 1020
-83-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
aacttagacg gttcaccagc aaacatcttt gaaggtggag aagctacttc tactacaggt 1080
aaactagctg ttggcattaa gcagggtgac tacaaagtag aagtacaagt tacaaaacgc 1140
ggtggtttaa cagtttctaa cactggtatt attacagtga aaaaccttga tacaccagct 1200
tctgcaatta aaaatgttgt atttgcatta gatgctgata atgatggtgt tgtaaactat 1260
ggcagcaagc tttctggtaa agactttgct ttaaatagcc aaaacttagt tgttggtgaa 1320
aaagcatctc ttaataaatt agttgctaca attgctggag aagataaagt agttgatcca 1380
ggatcaatta gcattaaatc ttcaaaccac ggtattattt ctgtagtaaa taactacatt 1440
actgctgagg ctgctggtga agctacactt actattaaag taggtgacgt tacaaaagac 1500
gttaaattta aagtaacgac tgattctcgt aaattagtat cagtaaaagc taacccagat 1560
aaattacaag ttgttcaaaa taaaacatta cctgttacat tcgtaacaac tgaccaatat 1620
ggcgatccat ttggtgctaa cacagctgca attaaagaag ttcttccgaa aacaggtgta 1680
gttgcagaag gtggattaga tgtagtaacg actgactctg gttcaatcgg tacaaaaaca 1740
attggtgtta caggtaatga cgtaggcgaa ggtacagttc acttccaaaa cggtaatggt 1800
gctactttag gttcattata tgtgaacgta acagagggta acgttgcatt taaaaacttt 1860
gaacttgtat ctaaagtagg tcaatatggc caatcacctg atacaaaact tgacttaaat 1920
gtttcaacta ctgttgaata tcaattatct aagtacactt cagatcgcgt atactctgat 1980
cctgaaaact tagaaggtta tgaagttgaa tctaaaaatc tagctgtagc tgacgctaaa 2040
attgttggaa ataaagttgt tgttacaggt aaaactccag gtaaagttga tatccactta 2100
acgaaaaatg gtgcaactgc tggtaaagcg acagtcgaaa tcgttcaaga gacaattgct 2160
attaaatctg taaacttcaa accagttcaa acagaaaact ttgttgagaa gaaaatcaac 2220
atcggtactg tattagagct tgagaagagt aacctggatg atatcgtaaa aggtattaac 2280
ttaacgaaag aaacacaaca taaagtacgt gttgtgaaat ctggtgcaga gcaaggtaaa 2340
ctttacttag atagaaacgg tgatgctgta tttaacgctg gcgatgtaaa acttggcgat 2400
gtaacagtat ctcaaacaag tgattctgca cttccaaact tcaaggcaga tctttatgat 2460
actttaacta ctaagtacac tgacaaaggt acattagtat tcaaagtatt aaaagataaa 2520
gatgttatta caagcgaaat cggttcacaa gctgtacacg tgaacgttct taataaccca 2580
aatctataa 2589
B. anthracis EA2 - (P49051, Q612R3, Q6KWJ4)
26. SQ SEQUENCE 814 AA; 86621 MW; C1638D26A1C6B101'CRC64;
MAKTNSYKKV IAGTMTAAMV AGVVSPVAAA GKTFPDVPAD HWGIDSINYL VEKGAVKGND
KGMFEPGKEL TRAEAATMMA QILNLPIDKD AKPSFADSQG QWYTPFIAAV EKAGVIKGTG
NGFEPNGKID RVSMASLLVE AYKLDTKVNG TPATKFKDLE TLNWGKEKAN ILVELGISVG
TGDQWEPKKT VTKAEAAQFI AKTDKQFGTE AAKVESAKAV TTQKVEVKFS KAVEKLTKED
IKVTNKANND KVLVKEVTLS EDKKSATVEL YSNLAAKQTY TVDVNKVGKT EVAVGSLEAK
TIEMADQTVV ADEPTALQFT VKDENGTEVV SPEGIEFVTP AAEKINAKGE ITLAKGTSTT
VKAVYKKDGK VVAESKEVKV SAEGAAVASI SNWTVAEQNK ADFTSKDFKQ NNKVYEGDNA
YVQVELKDQF NAVTTGKVEY ESLNTEVAVV DKATGKVTVL SAGKAPVKVT VKDSKGKELV
SKTVEIEAFA QKAMKEIKLE KTNVALSTKD VTDLKVKAPV LDQYGKEFTA PVTVKVLDKD
GKELKEQKLE AKYVNKELVL NAAGQEAGNY TVVLTAKSGE KEAKATLALE LKAPGAFSKF
EVRGLEKELD KYVTEENQKN AMTVSVLPVD ANGLVLKGAE AAELKVTTTN KEGKEVDATD
-84-
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WO 2007/044607 PCT/US2006/039293
AQVTVQNNSV ITVGQGAKAG ETYKVTVVLD GKLITTHSFK VVDTAPTAKG LAVEFTSTSL
KEVAPNADLK AALLNILSVD GVPATTAKAT VSNVEFVSAD TNVVAENGTV GAKGATSIYV
KNLTVVKDGK EQKVEFDKAV QVAVSIKEAK PATK
25. SQ Sequence 2445 BP; 974 A; 381 C; 479 G; 611 T; 0 other; 1260040913
CRC32;
atggcaaaga ctaactctta caaaaaagta atcgctggta caatgacagc agcaatggta 60
gcaggtgttg tttctccagt agcagcagca ggtaaaacat tcccagacgt tcctgctgat 120
cactggggaa ttgattctat taactactta gtagaaaaag gcgcagttaa aggtaacgac 180
aaaggaatgt tcgagcctgg aaaagaatta actcgtgcag aagcagctac aatgatggct 240
caaatcttaa acttaccaat cgataaagat gctaaaccat ctttcgctga ctctcaaggc 300
caatggtaca ctccattcat cgcagctgta gaaaaagctg gcgttattaa aggtacagga 360
aacggctttg agccaaacgg aaaaatcgac cgcgtttcta tggcatctct tcttgtagaa 420
gcttacaaat tagatactaa agtaaacggt actccagcaa ctaaattcaa agatttagaa 480
acattaaact ggggtaaaga aaaagctaac atcttagttg aattaggaat ctctgttggt 540
actggtgatc aatgggagcc taagaaaact gtaactaaag cagaagctgc tcaattcatt 600
gctaagactg acaagcagtt cggtacagaa gcagcaaaag ttgaatctgc aaaagctgtt 660
acaactcaaa aagtagaagt taaattcagc aaagctgttg aaaaattaac taaagaagat 720
atcaaagtaa ctaacaaagc taacaacgat aaagtactag ttaaagaggt aactttatca 780
gaagataaaa aatctgctac agttgaatta tatagtaact tagcagctaa acaaacttac 840
actgtagatg taaacaaagt tggtaaaaca gaagtagctg taggttcttt agaagcaaaa 900
acaatcgaaa tggctgacca aacagttgta gctgatgagc caacagcatt acaattcaca 960
gttaaagatg aaaacggtac tgaagttgtt tcaccagagg gtattgaatt tgtaacgcca 1020
gctgcagaaa aaattaatgc aaaaggtgaa atcactttag caaaaggtac ttcaactact 1080
gtaaaagctg tttataaaaa agacggtaaa gtagtagctg aaagtaaaga agtaaaagtt 1140
tctgctgaag gtgctgcagt agcttcaatc tctaactgga cagttgcaga acaaaataaa 1200
gctgacttta cttctaaaga tttcaaacaa aacaataaag tttacgaagg cgacaacgct 1260
tacgttcaag tagaattgaa agatcaattt aacgcagtaa caactggaaa agttgaatat 1320
gagtcgttaa acacagaagt tgctgtagta gataaagcta ctggtaaagt aactgtatta 1380
tctgcaggaa aagcaccagt aaaagtaact gtaaaagatt caaaaggtaa agaacttgtt 1440
tcaaaaacag ttgaaattga agctttcgct caaaaagcaa tgaaagaaat taaattagaa 1500
aaaactaacg tagcgctttc tacaaaagat gtaacagatt taaaagtaaa agctccagta 1560
ctagatcaat acggtaaaga gtttacagct cctgtaacag tgaaagtact tgataaagat 1620
ggtaaagaat taaaagaaca aaaattagaa gctaaatatg tgaacaaaga attagttctg 1680
aatgcagcag gtcaagaagc tggtaattat acagttgtat taactgcaaa atctggtgaa 1740
aaagaagcaa aagctacatt agctctagaa ttaaaagctc caggtgcatt ctctaaattt 1800
gaagttcgtg gtttagaaaa agaattagat aaatatgtta ctgaggaaaa ccaaaagaat 1860
gcaatgactg tttcagttct tcctgtagat gcaaatggat tagtattaaa aggtgcagaa 1920
gcagctgaac taaaagtaac aacaacaaac aaagaaggta aagaagtaga cgcaactgat 1980
gcacaagtta ctgtacaaaa taacagtgta attactgttg gtcaaggtgc aaaagctggt 2040
gaaacttata aagtaacagt tgtactagat ggtaaattaa tcacaactca ttcattcaaa 2100
gttgttgata cagcaccaac tgctaaagga ttagcagtag aatttacaag cacatctctt 2160
aaagaagtag ctccaaatgc tgatttaaaa gctgcacttt taaatatctt atctgttgat 2220
-85-
CA 02625349 2008-04-07
WO 2007/044607 PCT/US2006/039293
ggtgtacctg cgactacagc aaaagcaaca gtttctaatg tagaatttgt ttctgctgac 2280
acaaatgttg tagctgaaaa tggtacagtt ggtgcaaaag gtgcaacatc tatctatgtg 23.40
aaaaacctga cagttgtaaa agatggaaaa gagcaaaaag tagaatttga taaagctgta 2400
caagttgcag tttctattaa agaagcaaaa cctgcaacaa aataa 2445
While the invention has been described and illustrated with reference to
certain
embodiments thereof, those skilled in the art will appreciate that various
changes, modifications
and substitutions can be made therein without departing from the spirit and
scope of the
invention. For example, effective dosages other than the dosages as set forth
herein may be
applicable as a consequence of variations in the responsiveness insect
population beingtreated.
Likewise, the specific biochemical responses observed may vary according to
and depending on
the particular active compound selected or whether there are present
pharmaceutical carriers, as
well as the type of formulation and mode of administration employed, and such
expected
variations or differences in the results are contemplated in accordance with
the objects and
practices of the present invention. All references referred to herein are
incorporated by reference
in their entireties. The disclosures of the publications, patents, or patent
applications referred to
herein are hereby incorporated by reference in their entireties.
-86-
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